Muscular fatigue is a complex phenomenon affected by muscle fiber type and several metabolic and ionic changes within myocytes. Mitochondria are the main determinants of muscle oxidative capacity which is also one determinant of muscle fatigability. By measuring the concentrations of intracellular stores of high-energy phosphates it is possible to estimate the energy production efficiency and metabolic recovery of the muscle. Low intrinsic aerobic capacity is known to be associated with reduced mitochondrial function. Whether low intrinsic aerobic capacity also results in slower metabolic recovery of skeletal muscle is not known. Here we studied the influence of intrinsic aerobic capacity on in vivo muscle metabolism during maximal, fatiguing electrical stimulation.31P-Magnetic resonance spectroscopy (31P-MRS) combined with muscle force measurement from repeated isometric twitches.Animal subjects were genetically heterogeneous rats selectively bred to differ for non–trained treadmill running endurance, low capacity runners (LCRs) and high capacity runners (HCRs) (n = 15–19). We measured the concentrations of major phosphorus compounds and force parameters in a contracting triceps surae muscle complex using Our results demonstrated that phosphocreatine re-synthesis after maximal muscle stimulation was significantly slower in LCRs (p<0.05). LCR rats also became promptly fatigued and maintained the intramuscular pH poorly compared to HCRs. Half relaxation time (HRT) of the triceps surae was significantly longer in LCRs throughout the stimulation protocol (p≤0.05) and maximal rate of torque development (MRTD) was significantly lower in LCRs compared to HCRs from 2 min 30 s onwards (p≤0.05).We observed that LCRs are more sensitive to fatigue and have slower metabolic recovery compared to HCRs after maximal muscle contractions. These new findings are associated with reduced running capacity and with previously found lower mitochondrial content, increased body mass and higher complex disease risk of LCRs. This in turn has an important contribution to the force decline, especially with repeated maximal muscle stimulation Muscular fatigue is characterized with decline of force production caused by prolonged activation of skeletal muscle. It is known that muscle fiber types, characterized by differences in myosin heavy chain (MHC) composition, vary in force-generating capacity and resistance to fatigue In addition to muscle fiber type composition and myocellular metabolic and ionic changes the capacity to resist fatigue is determined by muscle oxidative capacity 31P-Magnetic resonance spectroscopy (31P-MRS) it is possible to measure the concentrations of the major phosphorus compounds in a contracting muscle, i.e., PCr and Pi e.g. exercise training) with aging.With Recent studies reveal that low aerobic capacity is associated with various metabolic and morphological properties in skeletal muscle, which in turn have pronounced effects on whole body level in health and disease in vivo during maximal, fatiguing stimulation in non-trained LCR/HCR rats. We hypothesize that low intrinsic aerobic capacity is associated with fatigue sensitivity, slow metabolic recovery and poor intramuscular pH homeostasis. We also hypothesize, that compared to HCRs, the twitch parameters of LCRs are more typical of fast twitch muscles due to larger relative amount of type 2b muscle fibers Since intrinsic aerobic capacity has pronounced effects in health, complex disease risk and life expectancy −1, increased 1 m min every 2 min) when the rats where 11 weeks of age . Rats were subsequently shipped to University of Jyväskylä when 16 weeks old. For experiments described here, 34 female HCR and LCR rats  weighing 194–332 g were used. Rats were from the 23rd generation of selection and 8 months of age when studied. Rats were housed 2/cage in an environmentally controlled facility  and received water and standard feed ad libitum.The HCR/LCR contrasting rat model system was produced via two-way artificial selection, starting from a founder population of genetically heterogeneous rats (N:NIH stock) in 1996, as described previously This study was approved by the National Animal Experiment Board, Finland (Permit number ESAVI-2010-07989/Ym-23). The 31P-MRS acquisition protocol was performed under isoflurane anesthesia.2 and 70% N2O. The right lower hindlimb was shaved and conductive electrolyte gel was applied at the heel and knee levels to optimize electrical stimulation of triceps surae muscle complex. Each rat was placed in a home-built cradle designed for non-invasive functional investigation of the right triceps surae. The cradle had a built-in strain-gauge sensor  and two transcutaneous electrodes to elicit and measure maximal twitch responses under isometric conditions. The foot was positioned on the pedal and the lower hind limb was centered on a flat radio frequency (RF) coil used for MRS measurement . The facemask was connected to open-circuit gas anesthesia equipment. During anesthesia, animal body temperature was maintained with a heated pad.The non-invasive MRS investigation setup was modified from the protocol designed by Giannesini et al. surement . The ped1H- and 20-mm-diameter 31P-coil) was used. The triceps surae muscle complex was carefully placed in the center of the coil combination, and the volume on coil was shimmed using 1H nuclear magnetic resonance (NMR) signal followed by pilot images with gradient echo pulse sequence to ensure that the position of the muscle was correct. 31P-MR spectra from the triceps surae were continuously acquired with pulse-acquire technique using 130 µs long, 90 degree block pulse for excitation, 1.855 s time to repeat, 4 kHz bandwidth covered with 2000 points. Prior to stimulation, a baseline spectrum was collected  and during the stimulation and recovery, a total of 15 spectra were acquired, resulting in temporal resolution of 56 seconds. Areas of PCr and Pi were obtained by a time-domain fitting routine using the AMARES-MRUI Fortran code Investigations were performed in a 4.7 T horizontal magnet . A flat surface transmit/receive RF coil (26-mm-diameter −1) during the time-course of transcutaneous electrical stimulation. A linear trend line was adjusted over 100 twitch responses in individual time-intervals that showed rapid linear reduction in the analyzed variables.The stimulation protocol consisted of baseline (8 min), stimulation (6 min) and recovery (8 min) measurements. Repeated isometric muscle twitches were elicited through electrical stimulation of triceps surae muscle complex at a frequency of 3.33 Hz . Force signal from the strain-gauge sensor was amplified, converted to digital signals by a 32-bit analog to digital converter , and processed using dedicated software . From each individual twitch response, 1) maximal force, 2) maximal rate of torque development (MRTD) and 3) half-relaxation time (HRT) were analyzed throughout the stimulation period. The twitch properties were then averaged over 15 second epochs . In addition, twitch force and MRTD values were normalized with their maximum value during the stimulation to compute relative rate of reduction . Statistical analyses for all variables were carried out using SPSS for Windows 13.0 statistical software . The Shapiro-Wilk test was used to investigate within group normality for a given parameter of interest. Levene's test was conducted to assess the homogeneity of the variance assumption. When assumptions were met independent-samples t-test was used. A statistical comparison of body mass and mean distance run to exhaustion was made using independent-samples t-test. The recovery of PCr level was compared with independent-samples t-test by comparing the individual slope values. Since the normality or equality of variance assumptions were not met, statistical comparisons of other studied parameters between LCR and HCR groups were made using Mann-Whitney test by comparing LCRs and HCRs at every time point. The twitch property values from the first last epochs  were alsversus 2093±25 m for HCRs (p<0.001), representing a 586% difference in running distance.LCR rats were 26% heavier compared to HCRs, the mean body mass of LCRs was 289±7 g and for HCRs 229±5 g (p<0.001). The mean distance run at exhaustion was 305±4 m for LCRs 31P-MRS results demonstrated that PCr resynthesis after stimulation was significantly slower in LCRs when comparing the slope values of linear trend lines adjusted between time points 7–9 min (p<0.05) ((p<0.05) . In bothPi levels increased similarly in both rat strains during stimulation period and reached the highest level after 4 minutes of stimulation . During LCRs maintained the intramuscular pH more poorly compared to HCRs . LCRs haMean twitch force curves from the first epoch (0–15 s) and from the last epoch (345–360 s) are presented in −1) in the beginning of the stimulation protocol than HCRs , although this trend was not significant. Later during the stimulation, LCRs showed a significantly lower twitch force from 2 min 45 s onwards compared to HCRs (p≤0.05) . Similarhan HCRs , but thihan HCRs  of the tThe rate of reduction in the normalized twitch force and MRTD during transcutaneous electrical stimulation occurred more rapidly in LCRs compared to HCRs (p<0.05) when the slope of individual twitch responses were compared .in vivo. LCRs have also slower metabolic recovery after continued contractions compared to HCRs.In the present study we examined noninvasively the characteristics of triceps surae muscle complex metabolism in two genetically heterogeneous rat strains (HCR/LCR) that widely differ for intrinsic (i.e. non-trained) aerobic capacity. Consistent with a lower maximal treadmill running capacity, we found that the skeletal muscle in LCRs become promptly fatigued during maximal muscle stimulation et al in turn found that the most significant differences in mitochondrial content between these rat strains were in white muscle type We hypothesized, that LCRs would have slow metabolic recovery, which is one sign of poor mitochondrial function and/or capacity 31P-MRS investigation when using surface coils. Another advantage of the isometric fatigue protocol is the constant muscle length that ensures measured force parameters are not affected by change in the muscle length. The non-invasive muscle stimulation method has been proven to stimulate solely the triceps surae muscle complex and not the antagonist muscles. In fact, Giannesini et al (2005) found in their study, using T2-weighted MR images, that the transcutaneous stimulation system activates specifically the gastrocnemius muscle of triceps surae A novel finding from the present study was that LCRs become promptly fatigued during maximal isometric muscle stimulation , not onlet al also showed that LCRs have significantly higher activation of the glycolytic enzyme phosphofructokinase Muscle fibers vary in force-generating capacity, contraction speed and resistance to fatigue et al demonstrated that HCRs maintain a higher degree of functional coupling between creatine kinase and adenine nucleotide translocase compared to the LCRs in slow-twitch muscle type It has been shown that HCR and LCR rats have similar resting muscle phosphocreatine, glycogen and ATP contents et al2PO4−) affect force production only at the end of the stimulation period. This suggested an indirect, time-dependent effect of these parameters on force production, which might be mediated by an alteration of Ca2+ fluxes throughout the sarcoplasmic reticulum (SR) 2+ release from SR occurs later in muscle fibers with high capacity for oxidative metabolism 2+ release due to higher oxidative capacity in skeletal muscle In skeletal muscle, the fatiguing stimulation resulted in depletion of PCr, accumulation of Pi, and a decrease in pH. As we hypothesized, LCRs had poor intramuscular pH homeostasis compared to HCRs. The larger contractile-mediated decrease in pH in the triceps surae of LCRs was likely related to the larger glycolytic capacity in fast-twitch muscle fibers (type 2b) of LCRs when compared to HCRs 2PO4− or Ca2+ fluxes, can also increase the half- relaxation time We hypothesized, that LCRs might have higher maximal twitch force (N) compared to HCRs due to bigger muscle size and larger relative amount of 2b muscle fibers with better force-generating capacity compared to 2a fibers of HCRs 31P-MRS measures of contractile function as reported here will be of substantial experimental value for studying the complexities of aging.The possible link between muscle oxidative capacity and metabolic diseases is an interesting topic. We have already shown that low aerobic capacity is related to higher complex disease risk


We determined the replicative capacity (RC) of these Gag-MJ4 chimeras by infecting the GXR25 cell line and quantifying virion production in supernatants via a radiolabeled reverse transcriptase assay. We observed a statistically significant positive correlation between RC conferred by the transmitted Gag sequence and set point VL in newly infected individuals (p = 0.02). Furthermore, the RC of Gag-MJ4 chimeras also correlated with the VL of chronically infected donors near the estimated date of infection (p = 0.01), demonstrating that virus replication contributes to VL in both acute and chronic infection. These studies also allowed for the elucidation of novel sites in Gag associated with changes in RC, where rare mutations had the greatest effect on fitness. Although we observed both advantageous and deleterious rare mutations, the latter could point to vulnerable targets in the HIV-1 genome. Importantly, RC correlated significantly (p = 0.029) with the rate of CD4+ T cell decline over the first 3 years of infection in a manner that is partially independent of VL, suggesting that the replication capacity of HIV-1 during the earliest stages of infection is a determinant of pathogenesis beyond what might be expected based on set point VL alone.Initial studies of 88 transmission pairs in the Zambia Emory HIV Research Project cohort demonstrated that the number of transmitted HLA-B associated polymorphisms in Gag, but not Nef, was negatively correlated to set point viral load (VL) in the newly infected partners. These results suggested that accumulation of CTL escape mutations in Gag might attenuate viral replication and provide a clinical benefit during early stages of infection. Using a novel approach, we have cloned  gag gene from 149 newly infected individuals from linked transmission pairs into a clade C proviral vector and determined the replicative capacity in vitro. We found that the replicative capacity conferred by the transmitted Gag correlated with set point viral loads in newly infected individuals, as well as with the viral load of the transmitting partner, and we identified previously unrecognized residues associated with increasing and decreasing replicative capacity. Importantly, we demonstrate that transmitted viruses with high replicative capacity cause more rapid CD4+ decline over the first three years, independent of viral load. This suggests that the trajectory of pathogenesis may be affected very early in infection, before adaptive immunity can respond.In the majority of HIV-1 cases, a single virus establishes infection. However, mutations in the viral genome accumulate over time in order to avoid recognition by the host immune response. Certain mutations in the main structural protein, Gag, driven by cytotoxic T lymphocytes are detrimental to viral replication, and we showed previously that, upon transmission, viruses with higher numbers of escape mutations in Gag were associated with lower early set point viral loads. We hypothesized that this could be attributed to attenuation of the transmitted virus. Here, we have cloned the  Despite a diverse HIV-1 quasispecies within chronically infected individuals, a single variant establishes infection in the majority of heterosexual transmission cases, resulting in a severe genetic bottleneck et al., manuscript in submission).Previous studies in both heterosexual and homosexual transmission pairs have demonstrated that viral loads (VL) in the newly infected partners are correlated with VL in the transmitting partner in vivo advantage for the virus, if a mutation occurs within a functionally constrained region of the genome such as Gag, it could reduce in vitro replicative fitness in vivo fitness benefit outweighs that of the replication cost It is clear that both humoral gag-pro sequences that confer reduced in vitro replicative capacities (RC) in vitro RC conferred by gag-pro variants isolated from both subtype B and C chronically infected individuals correlates to VL, demonstrating the role of intrinsic viral characteristics in defining this marker of pathogenesis Initial studies in the ZEHRP cohort of 88 transmission pairs demonstrated that the number of transmitted HLA-B associated polymorphisms in Gag, but not Nef, was negatively correlated to set point VL in the newly infected partners, suggesting that an accumulation of escape mutations might attenuate viral replication and provide a clinical benefit during early stages of infection in vitro RC conferred by the transmitted Gag sequence defines early clinical parameters of HIV-1 pathogenesis.The identification of 149 heterosexual epidemiologically linked transmission pairs from a discordant couple cohort in Lusaka, Zambia, provides a unique opportunity to investigate the role that HLA-mediated adaptation of Gag within a chronically infected individual plays in modulating the RC of the transmitted variant. We hypothesize that HLA-mediated adaptation of HIV-1 resulting in Gag variants conferring varying levels of RCs will be a major viral characteristic linking donor and recipient VLs, and that the gag sequences from the earliest seroconversion plasma sample from 149 newly infected recipients of epidemiologically linked Zambian transmission pairs into the MJ4 proviral backbone gag gene influences critical aspects of HIV-1 pathogenesis.To test this hypothesis and using a novel approach, we cloned gp41 sequences from both partners et al.All participants in the Zambia Emory HIV Research Project (ZEHRP) discordant couples cohort in Lusaka, Zambia were enrolled in human subjects protocols approved by both the University of Zambia Research Ethics Committee and the Emory University Institutional Review Board. Prior to enrollment, individuals received counseling and signed a written informed consent form agreeing to participate. The subjects selected from the cohort were initially HIV-1 serodiscordant partners in cohabiting heterosexual couples with subsequent intracouple  HIV-1 transmission Early set point VL for newly infected individuals was defined as the earliest stable nadir VL value measured between 3 and 9 months post infection and which did not show a significant increase in value within a 3–4 month window. HIV plasma VL was determined at the Emory Center for AIDS Research Virology Core Laboratory using the Amplicor HIV-1 Monitor Test . CD4+ T cell counts were based on T-cell immunophenotyping, with assays done using the FACScount System  in collaboration with the International AIDS Vaccine Initiative.xl DNA Analyzer (Applied Biosystems).Genomic DNA was extracted from whole blood or buffy coats . HLA class I genotyping relied on a combination of PCR-based techniques, involving sequence-specific primers (Invitrogen) and sequence-specific oligonucleotide probes (Innogenetics), as described previously Gag-pol population sequences were generated using nested gene specific primers. Combined RT-PCR and first round synthesis was performed using SuperScript III Platinum One Step RT-PCR (Invitrogen) and 5 µL viral RNA template. RT-PCR and first round primers include GOF (forward) 5′ ATTTGACTAGCGGAGGCTAGAA 3′ and VifOR (RT-PCR and reverse) 5′ TTCTACGGAGACTCCATGACCC 3′. Second round PCR was performed using Expand High Fidelity Enzyme (Roche) and 1 µL of the first round PCR product. Nested second round primers include GIF (forward) 5′ TTTGACTAGCGGAGGCTAGAAGGA 3′ and VifIR (reverse) 5′ TCCTCTAATGGGATGTGTACTTCTGAAC 3′. Three positive amplicons per individual were pooled and purified via the Qiagen PCR purification kit (Qiagen). Purified products were sequenced by the University of Alabama at Birmingham DNA Sequencing Core. Sequence chromatograms were analyzed using Sequencher 5.0 (Gene Codes Corp.), and degenerate bases were denoted using the International Union of Pure and Applied Chemistry codes when minor peaks exceeded at least thirty percent of the major peak height.Viral RNA was extracted from 140 µL plasma samples using the Qiagen viral RNA extraction kit (Qiagen). gag sequences was determined by building a neighbor-joining tree using Geneious v5.5.7 (Biomatters Ltd.). The percent similarity between nucleotide and amino acid alignment was determined based on the output matrix from these neighbor-joining trees. In calculating the percent similarity between amino acid sequences, degenerate bases that resulted in non-synonymous changes and, thus, a mixture of amino acid residues, were translated as an “X”. When one of the amino acids comprising a mixture in the donor was found in the recipient Gag sequence, this was counted as a mismatch, making the average percent similarity reported between donor and recipient gag sequences a maximal estimate of percent mismatch.The percent similarity between donor and recipient population gag sequencing of all 149 transmission pairs gag amplicons for Gag-MJ4 chimera generation using Phusion Hot Start II polymerase (Fisher) and 1 µL of the first round PCR product. Nested second round primers include GagInnerF1 (forward) 5′ AGGCTAGAAGGAGAGAGATG 3′ and BclIDegRev2 (reverse) 5′ AGTATTTGATCATAYTGYYTYACTTTR 3′, which generate a gag amplicon starting from the initiation codon of gag and extending 142 nucleotides after the gag stop codon and into pro. The 5′ portion of the MJ4 long terminal repeat (LTR) was amplified using Phusion Hot Start II polymerase and the MJ4For1b (forward) 5′ CGAAATCGGCAAAATCCC 3′ and MJ4Rev (reverse) 5′ CCCATCTCTCTCCTTCTAGC 3′ primer set. In order to make the proper insert for cloning, the patient-specific gag and MJ4-LTR sequences were joined using a splice-overlap extension PCR utilizing the MJ4For1b (forward) and BclIRev (reverse) 5′ TCTATAAGTATTTGATCATACTGTCTT 3′ primer set. Joined splice-overlap-extension PCR products were gel purified using the Qiagen gel extraction kit (Qiagen). Purified Gag-LTR inserts and wild-type MJ4 vector . First round PCR products were generated as was previously described for the Viral stocks were generated by transfecting 1.5 µg purified proviral plasmid DNA into 293T cells (American Type Culture Collection) using the Fugene HD transfection reagent (Roche) according the manufacturer's protocol. Viral stocks were collected 72 hrs post transfection, clarified by low-speed centrifugation, and frozen at −80°C. The titer of each viral stock was determined by infecting TZM-bl cells (NIH AIDS Research and Reference Reagent Program) with 5-fold serial dilutions of virus in a manner previously described 2.Both 293T and TZM-bl cell lines were maintained in complete Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (HyClone Laboratories), 2 mM L-glutamine, and 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (Gibco) at 37°C and 5% CO5 GXR25 cells 33P-labeled reverse transcriptase assay. Based on values obtained for days 2–8, the optimal window for logarithmic growth for all viruses was determined to be between days 2 and 6, as by day 8 many high replicating viruses had exhausted target cells causing a flattening or decline of the replication curve. Therefore, log10-transformed slopes were calculated based on days 2, 4, and 6 for all viruses. Replication scores were generated by dividing the log10-transformed slope of the replication curve for each Gag-MJ4 chimera by the log10-transformed slope of wild-type MJ4. Two independent Gag-MJ4 chimera clones per acutely infected linked recipient were run in duplicate to confirm cloning fidelity. After both independent clones were confirmed to have identical replicative capacities, one clone was subsequently run in triplicate in two independent experiments in order to generate consistent replicative capacity scores. GXR25 cells were maintained in complete RPMI medium supplemented with 10% fetal bovine serum (HyClone Laboratories), 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate (Gibco), and 10 mM HEPES buffer at 37°C and 5% CO2.In order to assess the RC of Gag-MJ4 chimeras, 5×10Aliquots of culture supernatants from infected cells were added to an RT-PCR master mix gag sequences from Zambia and South Africa . Polymorphisms that increased or decreased replicative capacity were defined based on amino acid associations with RC derived from an exploratory pair-wise analysis detailed in the experimental results. To generate a summed polymorphism score, the number of HLA-associated fitness-decreasing polymorphisms was subtracted from the number of HLA-associated fitness-increasing polymorphisms.HLA-associated polymorphisms were defined as any non-consensus polymorphism that occurred at an amino acid position having known escape mutations adapted to specific HLA-class I alleles as defined by a list of associations generated in a manner similar to that described previously The relationships between RC and set point VL, donor VL, and average CD4+ counts; RC and the number and quality of HLA-associated polymorphisms; and VL and the number and quality of HLA-associated polymorphisms were analyzed using the Spearman rank correlation. Linear regression analyses were utilized to generate trend lines to facilitate visualization of correlation graphs. Mann-Whitney tests were used to compare the differences in RC between rare and more common polymorphisms. Mann-Whitney tests were used to analyze the difference in median set point VLs between different RC groups . All Spearman correlations, Mann-Whitney tests, and linear regression analyses were performed using Prism GraphPad v5.0 .The Mann-Whitney U test was used to identify statistically significant differences in RC between two groups (e.g. sequences with or without a given HIV polymorphism). Multiple tests were addressed using q-values 3. The set point VL is another factor being considered. Kaplan Meier curve and log-rank test were used to compare the survival between the groups with RC<1 and RC>2. Cox proportional hazard regression was used to assess risk associated with high RC with or without adjustment for the confounding factor of set point VL. The sample size (n = 66) was inadequate for a more complete analysis with additional covariates.A subset of volunteers with longitudinal CD4+ T cell counts (n = 63) was analyzed to characterize the relationship between RC and T cell decline. Survival analysis was used to estimate the association between replicative capacity (RC) and the drop of CD4+ cell count. The endpoint is defined as the time before CD4+ T cell counts drop below a threshold, e.g. 300 or 350 cells/mmStudying heterosexual transmission of HIV-1 within the context of discordant couples allows for full characterization of viruses from both donor and recipient. From a total of 294 epidemiologically linked transmission pairs identified from 1998–2010 in the Zambia Emory HIV Research Project (ZEHRP) discordant couple cohort 10 VL for donors near the time of transmission and the median log10 set point VL for linked recipients was 5.02 (IQR = 4.51–5.45) and 4.39 (IQR = 3.91–4.99) respectively for all participants included in this study. gag gene of the 149 epidemiologically linked transmission pairs and highlights the high degree of sequence similarity between donor and recipient viruses.The median loggag sequences isolated from acute/early time points of linked recipients were homogeneous and in most cases were identical to the donor population gag sequence isolated near the EDI. Overall, donor and linked recipient sequences differed in amino acid composition by only 2.7%. Mutations were counted when a mixture of nucleotides (amino acids) in the donor population sequence resolved to a single residue in the recipient and thus represent maximal values. Of the 149 pairs only 37 recipient sequences had evidence of potential early escape and a majority of these individuals (28/37) had only a single amino acid change. Therefore, we can conclude that the majority of the sequence polymorphisms present at the seroconversion time point are derived from the chronically infected donor. Additional characteristics of the cohort including set point VL and CD4+ counts are listed in The majority of population gag-deleted NL4-3, and resulting viruses propagated in permissive cells gag gene into a replication competent, CCR5 tropic, clade C provirus, MJ4 Previous studies investigating the role of Gag viral fitness have employed a recombination approach in which sequences are PCR amplified as a bulk population, allowed to recombine into a gag sequence. This ensures that the cis-acting sequences upstream of Gag, which may influence expression levels, are constant throughout all constructs. The resulting chimeras include the entire transmitted gag sequence from the initiation codon to the end of Gag and extend into conserved region of protease by 47 amino acids. For each newly infected individual, at least two independent Gag-MJ4 chimeras were sequence confirmed and assayed for replicative capacity (RC). An analysis of variation of RC between the two independent clones derived from each newly infected linked recipient was 8.5%. Testing two independent clones, therefore, ensures that the observed RC is not due to the confounding effect of backbone mutations that might have arisen during the cloning process and provides an estimate of experimentally induced variation.A splice-overlap-extension PCR was employed to fuse the MJ4-LTR-U5 sequence with the transmitted gag population sequences isolated from linked recipients, with only 28% having one or more mixed bases resulting in amino acid changes in the sequences from which the Gag-MJ4 chimeras were generated. When this was the case, the gag clone with the sequence closest to the donor gag sequence was chosen in order to avoid sampling gags containing de novo escape or reversion. In some cases in which multiple variants appeared to be transmitted, several gag variants were assayed for RC as described in the materials and methods section. In each case, these minor variants were found to have similar or identical RC values (data not shown).Overall, a low amount of heterogeneity was detected in In initial replication assays performed in order to test assay precision, wild-type MJ4 exhibited an intra-assay variability of 10.4% and an inter-assay variability of 8.7%. An examination of the RC of transmitted viruses allows us to determine the role of viral replication in defining set point VL in acutely infected individuals before significant viral adaptation to immune pressure of the host has taken place, which might confound the relationship of RC to VL. We observed a statistically significant positive correlation between the replicative capacities of Gag-MJ4 chimeras and set point VLs in newly infected individuals  and 4 residues at 3 unique positions that were significant at p<0.002 (q<0.2) (Uncovering sites of vulnerability in HIV-1 is a high priority for the informed design of an effective HIV vaccine  (q<0.2) . In whatThe locations of all statistically significant polymorphisms (p<0.05), along with their effects on RC as compared to the median RC of all viruses, are plotted linearly on a graphical representation of the Gag protein .. Residugag sequences from Zambia and South Africa  was utilized to identify residues that affected RC and were also HLA-associated. Within this data set, HLA-associated polymorphisms are classified as being either adapted or non-adapted. An adapted residue is one that is escaped relative to the HLA-allele in question. In contrast, a residue that is non-adapted is the susceptible form and may render the virus vulnerable to immunological targeting by the HLA-allele in question. This new dataset has identified a total of 199 HLA-linked polymorphisms  vs. 59 associations utilized previously from a smaller subset of gag sequences An expanded data set of 1899 subtype C During our analysis of amino acid polymorphisms linked to changes in RC (p<0.05), we observed a negative correlation between the frequency of polymorphisms and the magnitude of their effect on RC . Indeed, rare polymorphisms, those occurring in less than 10 of the 149 individuals studied, had significantly greater impact (both negative and positive) on RC than polymorphisms that occurred more frequently . This figag sequences from Zambia and South Africa , described above, was employed. The number of non-consensus polymorphisms located at HLA-associated positions was determined for each MJ4 chimera and then correlated with the RC defined by those Gag sequences. Surprisingly, we found a positive association between the number of HLA-associated polymorphisms and RC  for whom CD4+ T cell counts were available at regular three month intervals for greater than one year post-infection.3  and those infected with highly replicating viruses (RC>2), within the first 3 years of infection  within each linked pair, allowing us to conclude that the majority of polymorphisms in Gag present at the seroconversion time point were transmitted from the chronically infected donor.In this study of 149 linked Zambian heterosexual transmission pairs from the ZEHRP discordant couple cohort we have more fully characterized the role that HIV-1 viral characteristics, in particular the replicative capacity (RC) conferred by the transmitted gag genes from acutely-infected recipients into a primary isolate (MJ4) provirus has many important advantages over previously employed methods. MJ4 is a CCR5 tropic infectious molecular clone derived from a subtype C clinical isolate from Botswana, providing greater homology to viruses circulating within the Zambian population than other previously used subtype B lab-adapted strains gag stop codon in MJ4 does result in a chimeric protease, however, this region is 96.5% conserved in this cohort and we did not observe a high prevalence of dead or inactive Gag-MJ4 chimeras.Since all of the transmission pairs in this study were infected with subtype C viruses, our approach of precisely cloning gag sequences into MJ4 on virus replication was highly significant, with many of the chimeras exhibiting RC values greater than a hundred-fold higher than wild-type MJ4, which in this assay is one of the poorer replicators. This indicates that substitution of Gag can drastically alter the in vitro RC of the virus when all other viral components are constant. Multiple intra-molecular contacts as well as host protein interactions in Gag are necessary for effective intracellular Gag trafficking The impact of engineering foreign et al., which have demonstrated a statistically significant link between the RC conferred by gag-pro genes in subtype B and C chronically infected individuals to VL It has been well established that the set point VL in those recently infected with HIV-1 is correlated to disease progression and clinical outcome gag sequence and the early set point VL in newly infected individuals. This result implies that RC plays a role in defining the overall level of virus replication during the first year of infection. Moreover, in multivariable analyses that take into account the early viral control imposed by the B*57 allele and by gender, the impact of RC on set point VL was found to be independent of these two host factors (p = 0.009). Other factors such as NK cells and restriction factors such as TRIM or APOBEC may potentially affect RC and VL, however little is currently known regarding these potential effects, and future efforts should evaluate the role of such factors. While we observed a statistically significant positive correlation between RC and set point VL, outliers in the data exist that do not fit the trend, and in some cases can be explained by the presence of protective HLA-alleles or by a large number of escape mutations present in the transmitted sequence that are relevant to the HLA background of the newly infected individual. Set point VL is clearly determined by a combination of both host factors, including HLA-alleles, and viral factors such as RC, and this may explain the differences in the absolute correlation for each individual.In contrast, in this large group of very early ZEHRP seroconvertors (with samples drawn a median of 45 days post-EDI) we observed a clear statistically significant correlation (p = 0.02) between the RC conferred by the transmitted The RC of Gag-MJ4 chimeras also correlated with VLs near the estimated date of infection in chronically infected donors, consistent with the previously reported observation that donor and recipient VLs are correlated within epidemiologically linked transmission pairs In a pair-wise analysis, a large number of residues were associated with changes in fitness , with 4 residues at 3 unique positions at q<0.2. These residues included the polymorphisms 30R and 31I in p17 (MA), and 309S in p24 (CA). However, in an exploratory analysis of those residues associated with changes in RC with a p value<0.05, it was clear that associated polymorphisms were noticeably enriched on a per residue basis in p17 and p2 . The former plays critical roles in intracellular trafficking, and membrane association of Gag Polymorphisms positively or negatively affecting replication in the p24 region of Gag were limited to just six residues , in accordance with the conserved nature of this protein. Surprisingly, none of the canonical B*57/B*5801 associated escape mutations within p24, whose fitness defects have been well documented in vitro impact of mutations at residue 12 on replication is supported by a longitudinal study of a subset of this seroconvertor cohort (n = 81), in whom polymorphisms at residue 12 were found to revert at a high rate (25%/yr), over the first two years of infection . Furthermore, in this same study, escape at position 12 occurred only once and at 24 months post-infection in a total of ten A*74 positive individuals, confirming the high fitness cost associated with CTL-induced escape mutations at this position. We hypothesize that the targeting of this putative epitope, KR9 The most deleterious HLA-associated mutation that we observed was K12E, which reduced RC by almost 10-fold relative to the median RC of the cohort. This polymorphism is found quite rarely in the population (3 out of 149), and is statistically associated with HLA-A*74, an allele found to be highly protective in both this Zambian subtype C cohort as well as others Rare mutations, such as K12E, which occur in a small subset of the population studied here , affected fitness to a statistically greater degree than more common polymorphisms. Rare fitness decreasing mutations are likely unique to specific circumstances such as those where a considerable decrease in RC is warranted in the face of a very effective cellular immune response that is largely abrogated upon mutation. Such mutations have been found to subsequently revert after transmission to individuals lacking the selecting HLA-allele in vivo fitness cost that cannot be captured in the in vitro replication system used here. Due to the fact that these mutations are rare, they are difficult to statistically link to HLA alleles or to link to other residues with which they may covary, making the potential fitness defects that these mutations mitigate difficult to elucidate.A similar observation was made for rare mutations that greatly increase RC. Global compensatory mutations do exist that can compensate multiple deleterious mutations, such as those within the cyclophilin binding loop et al., manuscript in preparation) we report a weak positive correlation between the total number of HLA-associated polymorphisms in Gag and RC. The fact that this correlation was positive is consistent with our observation that a large fraction of the non-consensus HLA-associated polymorphisms increased RC. In particular, in the expanded data set of HLA-associated polymorphisms, we observed that non-adapted residues, which would be predicted to render the virus susceptible to the linked HLA allele, were statistically associated with increased fitness.A key goal of this study was to understand how the cellular immune response might select for mutations in Gag that reduce viral RC, and while identification of specific amino acid polymorphisms that either increase or decrease fitness can be informative, it is equally important to elucidate how the accumulation of specific HLA-associated polymorphisms in Gag affects both RC of the virus and VL in the newly infected person. Previous efforts to correlate the total number of HLA-associated polymorphisms in Gag to RC have yielded inconclusive results in vitro fitness. This is consistent with the findings of Kawashima et al.et al.gag-pro sequences most disparate from the subtype C consensus gag-pro sequence had statistically higher replicative capacities than their more consensus-like counterparts, and this finding has been recapitulated in this current study. Taken together, these data suggest that overall, HLA-mediated adaptation is driving the fixation of consensus residues that are less fit than their susceptible counter-parts.These findings suggest that CTL escape mutations, which decrease the overall RC of the virus, are being driven to consensus as a result of population level immune pressure. In the absence of immune pressure, the non-escaped (non-adapted) residues would be expected to predominate, but if they render the virus susceptible to a large portion of the population, then the consensus residue will be escaped rather than susceptible, despite reducing When we account for this ability of HLA-associated polymorphisms to either increase or decrease fitness by assigning a summed polymorphism score, which subtracts the number of fitness decreasing polymorphisms from the number of fitness increasing polymorphisms in a particular sequence, we find a highly statistically significant correlation between RC and the summed polymorphism score (p<0.0001). Although this p-value should be interpreted cautiously, since it reflects the summation of features previously identified to be correlated with RC, the data do suggest that the effect of polymorphisms is cumulative, and that as a Gag sequence accumulates an excess of fitness-reducing polymorphisms, the RC decreases proportionally. Similarly, utilization of a summed polymorphism score improved previously reported correlations between the total number of HLA-associated polymorphisms in Gag and set point VL in newly infected individuals de novo escape mutations. Consequently, we analyzed individuals with longitudinal CD4 counts out to three-years post infection in order to determine if the observed early benefit was sustained in early chronic stages of infection. Using Kaplan-Meier survival analyses to examine the relative time for individuals infected with viruses encoding gag genes conferring RC values of <1 and >2 to reach CD4 T cell counts of 350 after 3 years of infection, we observed a clear and statistically significant difference. This was even more striking when CD4 counts less than 300 were used as the endpoint. Moreover a Cox proportional hazard model demonstrated a significantly increased risk of CD4 counts falling below both 350 (HR 2.36) or 300 (HR 3.80) over the first three years of infection for individuals whose gag gene conferred an RC>2 vs. RC<1. These findings indicate that the RC conferred by the transmitted Gag sequence may have profound and prolonged effects on HIV-1 pathogenesis from acute to early chronic stages of infection.While VL has been demonstrated to influence the rate of disease progression in HIV-1 infected individuals While RC and VL are correlated in the full data set, set point VL does not fully explain the effect of RC on CD4, because we did not observe any statistically significant differences in set point VL between the two groups (RC<1 and RC>2) for the subset of individuals with CD4+T cell counts (n = 63). Moreover, in Cox proportional hazard models which take into account VL, the HR remained high (2.17 and 3.11 respectively) and p values continued to trend or remain borderline significant (p = 0.093 and 0.051). This suggests that both VL and RC have independent effects on CD4 decline, however, because this analysis was conducted on a subset of less than half of our initial cohort, additional work is underway to further confirm and extend these results.It seems possible, therefore, that the RC of the transmitted variant may initiate crucial events, early in infection and dissemination, that dictate both acute and later stage pathogenesis regardless of the ability of the immune system to control viral replication down to set point. Infection with highly replicating variants could lead to a more complete depletion of central memory CD4+ T cell pools at this early time that could predispose an individual to more rapid CD4+ T cell loss, irrespective of adequate control of viral replication. This is evidenced in a few individuals infected with highly replicating Gag variants (RC>2), who go on to control VL to a low set point, but whose CD4+ T cells counts rapidly drop below 300 (data not shown). Additionally, a high level of peak viremia or initial high antigen loads could establish an inflammatory environment that leads to sustained immune activation, which has been implicated as a more reliable marker for disease progression in vitro approach to define the impact of polymorphisms in Gag on transmitted virus RC has clearly shown that this property of the virus is a significant contributor to early set point VL in a newly infected individual. More importantly, however, these studies suggest a critical role for RC in defining the trajectory of immune depletion and pathogenesis, beyond simply its impact on VL, and highlight the importance of the very earliest events in virus-host interactions. It also raises the possibility that a vaccine that can attenuate early virus replication would have a positive impact both on vaccinated individuals, as well as non-vaccinated individuals by weakening the transmitted/founder virus and increasing the likelihood of transmission of low replicating variants.In summary, using an Figure S1Donor and recipient population gag sequences cluster with one another. Gag population sequences from donors and linked recipients were amplified and sequenced as described in the methods section. Nucleotide gag sequences were aligned using the Gene Cutter tool accessible on the Los Alamos Nation Lab HIV Sequence Database (http://www.hiv.lanl.gov/content/sequence/GENE_CUTTER/cutter.html) and a neighbor-joining tree was generated using the Geneious sequence analysis software v5.5.7 (Biomatters Ltd.). The radial tree was annotated using the Interactive Tree of Life (iTOL) online tool for phylogenetic tree display and annotation . Blue denotes transmitting partners (donors) and green denotes linked recipients. This tree demonstrates the high degree of similarity between donor and linked recipient gag population sequences within epidemiologically linked transmission pairs.(EPS)Click here for additional data file.Figure S2Gag sequences that are less like the Gag subtype C consensus sequence replicate more efficiently in vitro. The Gag amino acid sequences of all Gag-MJ4 chimeras were compared to a Zambian subtype C consensus Gag sequence  by building a neighbor-joining tree using the Geneious sequence analysis software v5.5.7 (Biomatters Ltd.) and the percent similarity to consensus was determined for each sequence. Notably, viruses encoding Gag sequences most disparate from the subtype C consensus Gag sequence replicated to higher levels, and we observed a statistically significant negative correlation between RC and the percent similarity of Gag to consensus .(EPS)Click here for additional data file.Table S1Amino acids in Gag associated with changes in replicative capacity. This table lists all amino acids associated with changes in RC. Residues that remain significantly associated with changes in RC after correction for multiple comparisons (q<0.2) are depicted in green. A total of 152 sequences and RC values were available for association analysis, with 149 of these with sufficient clinical follow-up for inclusion in the broader study. a ΔRC is defined as median RC of all viruses tested (∼1.5) subtracted from the median RC of all viruses with the particular polymorphism. b The location of epitopes was defined by the compendium of “A-list” epitopes available in the LANL Immunology Database (HIV Molecular Immunology 2009). c HLA class I alleles restricting epitopes harboring these polymorphisms that affect RC were also defined base on the LANL Immunology Database compilation of “A-list” epitopes.(DOC)Click here for additional data file.


Adoptive cell therapy (ACT) with expanded autologous tumor infiltrating lymphocytes (TIL) has emerged to be a powerful salvage therapy for metastatic melanoma with response rates up to 50%. However, the generation of the TIL ACT product is technically challenging with current methods requiring a large excess of allogeneic normal donor (pool) or patient-derived PBMC (200:1 ratio to the TIL) used as “feeders” to support a rapid expansion protocol (REP) to generate the final TIL infusion product. Because PBMC feeder products consist of a heterogenous cell population, it introduces undesired variability in TIL expansion rates and phenotype, especially in the yield of CD8+ T cells that are the most active component of the TIL product. Here, we have developed an alternative to PBMC feeders using a K562-based artificial antigen-presenting cell (aAPC) system expressing CD64, CD86, and 4-1BBL that is much more practical and cost-effective. Using PBMC feeders as controls, we found an optimal aAPC:TIL ratio (50:1) supporting maximal TIL expansion used for subsequent experiments. Analysis of the resulting TIL products from multiple experiments revealed that the aAPC induced higher rates of CD8+ T cell expansion with a comparable effector-memory phenotype as TIL expanded with the traditional PBMC feeder approach. The exceptions were CD56 that was more highly expressed on the CD8+ cells and CD28 which had a lower expression. Notably, TIL expanded with aAPC were enriched in CD8+ BTLA+ T cells, a subset highly correlated with clinical response to ACT. TCR Vαβ clonotype analysis also found that TIL expansion with the aAPC did not alter the diversity of the T cell repertoire. Further analysis using gene chip profiling revealed significant differences in gene expression in TIL products expanded using the aAPC versus with PBMC feeders. These included an up-regulation of certain genes in the Wnt pathway, cyclic nucleotide metabolism, and multiple genes in different stem cell pathways. This more stem-like profile may have beneficial properties following adoptive transfer. Finally, we found that CD8+ TIL expanded with aAPC had a similar anti-tumor effector cell activity in CTL and IFN-γ release assays. Overall, our data demonstrates that this aAPC system is a suitable alternative to generate clinical-grade melanoma TIL infusion products for ACT that produces comparable and perhaps even more active T cells than current methods. Our group is currently phasing in the use of these aAPC for our GMP TIL production.


Phocarctos hookeri) in two contrasting environments. The NZ sea lion is one of the rarest pinnipeds in the world. Most of the species is based at the subantarctic Auckland Islands , with a recolonizing population on the Otago Peninsula, NZ mainland . We investigated how juvenile NZ sea lions adjust their foraging behaviour in contrasting environments by deploying satellite-linked platform transmitting terminals (PTTs) and time-depth recorders (TDRs) on 2–3 year-old females at AI (2007–2010) and Otago (2009–2010). Juvenile female NZ sea lions exhibited plasticity in body size and behaviour. Otago juveniles were significantly heavier than AI juveniles. Linear mixed effects models showed that study site had the most important effect on foraging behaviour, while mass and age had little influence. AI juveniles spent more time at sea, foraged over larger areas, and dove deeper and longer than Otago juveniles. It is difficult to attribute a specific cause to the observed contrasts in foraging behaviour because these differences may be driven by disparities in habitat/prey characteristics, conspecific density levels or interseasonal variation. Nevertheless, the smaller size and increased foraging effort of AI juveniles, combined with the lower productivity in this region, support the hypothesis that AI are less optimal habitat than Otago. It is more difficult for juveniles to forage in suboptimal habitats given their restricted foraging ability and lower tolerance for food limitation compared to adults. Thus, effective management measures should consider the impacts of low resource environments, along with changes that can alter food availability such as potential resource competition with fisheries.Foragers can show adaptive responses to changes within their environment through morphological and behavioural plasticity. We investigated the plasticity in body size, at sea movements and diving behaviour of juvenile female New Zealand (NZ) sea lions ( Foraging behaviour can be influenced by numerous intrinsic  and extrinsic  factors Arctocephalus gazella) Individuals from populations that exploit lower quality habitats  may need to expend more effort foraging than conspecifics exploiting better quality habitats  provides an opportunity to examine plasticity in foraging behaviour of an understudied age class between contrasting environments, in a declining species of management concern. The NZ sea lion is one of the rarest and most highly localized pinnipeds  in the world th century The New Zealand (NZ) sea lion  Thyrsites atun, 6.1 kJ/g; jack mackerel, Trachurus sp., 7.6 kJ/g) These large disparities between the two populations of adult female NZ sea lions suggest the extreme foraging behaviour, smaller size and low milk fat content of AI females are not species-specific, but demonstrate plasticity in behaviour, morphology and physiology in response to differences in their environment. Prey species also differed between AI and Otago NZ sea lion diet Juveniles do not have as high energetic costs as lactating females This study was conducted with approval from the Department of Conservation (DOC) and University of Otago Animal Ethics Committees . Instrument deployments were performed under inhalant gas anaesthesia, and all efforts were made to minimize pain and suffering.We collected data over four seasons from January–February , 2007–2010 at Sandy Bay, Enderby Island in the NZ subantarctic AI and over two seasons from March–May , 2009–2010 at the Otago Peninsula, South Island, NZ . LogistiStudy animals included a sample of 2 and 3-year-old juvenile female NZ sea lions at AI and the entire known population of 2 and 3-year-old females at Otago. All animals were aged accurately based on flipper-tags that were attached within a few months after birth. We should note the logistical difficulties of deploying and recovering instruments on juveniles, a rarely seen age group , restricted our sample sizes at AI whereas study animals at Otago were limited by the small population size. We captured animals using a specially designed hoop net and physically restrained them with two handlers Sea lions were instrumented with satellite-linked time-depth recorders  or platform transmitting terminals  and time-depth recorders . Sea lions were also instrumented with very high frequency  transmitters to facilitate recaptures. Due to equipment availability, some AI individuals were only deployed with PTTs and VHFs. We attached the instruments to the dorsal pelage of the animal below the shoulder blades on the back midline using two-part epoxy resin. Once the instruments were securely attached, we stopped the flow of anaesthetic. We measured standard body length (nose to tail) while the sea lion was allowed to recover and observed each animal until it was fully conscious. We recaptured each animal before the end of the field season to retrieve instruments.Animal positions at sea were estimated by the Argos satellite system and assigned to different location classes based on their accuracy. However, there are large variations in accuracy of the various location classes As a result of the small spatial scale of the satellite data of Otago females and based on personal observations of foraging activities of sea lions within a few meters from shore during our study, a custom-designed algorithm based on Freitas et al. For AI females, we defined complete foraging trips as trips with the start and end locations within 10 km of Enderby Island TDRs sampled dive depth (±0.5 m) every 5 s when wet. Depth readings were corrected for shifts in the pressure transducer at the surface of the time-depth recorders before analyses. The dive profiles were visually examined for offsets from the surface and the zero-offset correction was performed using the offset method in the package diveMove We tested for mass differences between age and study site groups with t tests or Wilcoxon rank sum tests. We also derived a body condition index (BCI) from the residuals of a linear regression of mass against body length  [57]. StAlthough inter-annual variation may influence the foraging behaviour observed, small sample sizes each year precluded the differentiation of annual and individual differences. Furthermore, foraging studies on individual adult NZ sea lions followed across several years indicate little annual variability in foraging behaviour We centred and standardized the age and mass predictor variables to improve the interpretability of regression coefficients We assessed the primary productivity using average chlorophyll a concentration as a proxy  at the tBetween January–February 2008–2010, we retrieved satellite data for 19 females  and dive data for 12 females  at AI . BetweenP<0.01; P  = 0.20; P<0.01; 3-year-olds t = −5.7, df  = 12, P<0.01). Overall, Otago animals had better body condition than AI animals (P  = 0.53).The 3-year-old juvenile females were significantly heavier than 2-year-olds at AI  than the AI (0.43±0.12 mg/m3) for the duration of the study period . The yearly averages ranged from 0.41–0.45 mg/m3 at AI and 1.21–2.48 mg/m3 at Otago from 2007–2010.The average chlorophyll a concentration was significantly higher at the Otago Peninsula  when there is lower prey abundance around the central place The mass of AI study animals were within the range of mean mass calculated for 2 and 3-year-old female NZ sea lions in a larger study Arctocephalus fosteri) Although differences in habitat-related effects such as prey availability may influence the observed contrasts in juvenile NZ sea lion foraging behaviour, confounding factors such as differences in conspecific density levels or season may also impact foraging behaviour. For instance, AI individuals may face higher intraspecific competition because ∼10,000 NZ sea lions inhabit the NZ subantarctic islands Seasonal differences in prey distribution may also explain differences in foraging behaviour between AI and Otago individuals since data were collected in the austral summer and autumn, respectively. For example, prey may be distributed at deeper depths or farther locations during the summer and at shallower depths and closer to colonies during the autumn. Hence, it is possible that juvenile NZ sea lions may exhibit more similar foraging behaviour in the same season. However, diet studies of both populations in the autumn have found AI and Otago animals forage on different prey types in the same season Low food availability can have large impacts on population dynamics since unfavourable environmental conditions can have immediate effects on animal performance  Juvenile NZ sea lion foraging behaviour differed between study sites, with age and mass having little influence on foraging behaviour. Otago juvenile and adult female sea lions are larger and feed on higher energy prey Figure S1Relationship between mass and body length of juvenile (2 and 3-years-old) female New Zealand sea lions (Phocarctos hookeri) and a definition of the body condition index (BCI) as the residual value between observed and expected mass.(DOC)Click here for additional data file.Table S1Morphometric, dive and satellite data available for individual juvenile female New Zealand sea lions (Phocarctos hookeri) at Auckland Islands (AI) and Otago Peninsula. BCI, body condition index.(DOC)Click here for additional data file.Table S2Results of linear mixed effects models run on juvenile New Zealand sea lion (Phocarctos hookeri) foraging trip characteristics: at sea duration, trip distance and maximum distance from study site.(DOC)Click here for additional data file.Table S3Results of linear mixed effects models run on juvenile New Zealand sea lion (Phocarctos hookeri) dive characteristics: mean and maximum dive depth and mean and maximum dive duration.(DOC)Click here for additional data file.


Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid  and inexpensive for routine clinical use. In nature, microbes exist in complex communities shared with other species rather than as pure cultures dominating an ecological niche. The microbiota in healthy humans in vitro. Only a small fraction of all bacteria can be successfully cultured, while clinically significant organisms may be slow-growing, fastidious, inert, or unviable Traditional microbiological classification is rooted in organisms' morphology and biochemical properties and first requires that species are isolated by growth As first realized through metagenomics research Here, we develop a rapid and inexpensive culture-free next-generation sequencing assay able to accurately catalog bacterial species directly from highly complex patient specimens by 16S rRNA gene deep sequencing. As a proof of principle, we explore the utility of this assay in comparison to existing clinical microbiology techniques across a collection of challenging clinical samples and cystic fibrosis sputum samples.Although human-derived samples were used in this study, this work is not considered human subjects research, and is not considered to involve human participants per University of Washington Human Subjects Division because the material constituted non-identifiable, leftover clinical specimens that were not collected specifically for this study (UW IRB Doc #295). As this work is not human subjects research and does not involve human participants, this work is exempt from needing ethical approval and written informed consent:“Use of Non-Identifiable Specimens/Data . . .requires neither determination of exempt status nor IRB review” (UW IRB Doc #295).Per UW IRB Document #359:“2.4.2 The UW IRB interprets this definition to mean that a human specimen falls within the definition of health care information only when:It can be tied to an individual, andIt was obtained in the course of diagnosing, treating, or otherwise providing health care in the state of Washington.2.4.3 This means that a human specimen is not, in and of itself, considered to be a human subject by state law, unless it can be readily connected to an individual.”Only “If your research activity involves human subjects, it is necessary to complete the appropriate HSD form for submission, review, and approval prior to commencement of the research activity.”Use of leftover, non-identifiable patient samples is ‘Not Human Subjects Research’ as defined by the UW IRB, and does not require UW IRB approval (UW IRB Doc #295).We completed and filed with the UW IRB a self-determination form for approval of Use of Non-Identifiable Specimens/Data, which was approved by the Department Chair of Laboratory Medicine, and which states that “the project requires neither determination of exempt status nor IRB review” (UW IRB Doc #295).With specific respect to written informed consent: Per UW IRB review , this work did not involve human participants, and thus, no waiver for the need of written informed consent was required. The University of Washington IRB board has deemed this research “Not Human Subjects Research”, therefore does not involve human participants (UW IRB Doc #295).Microbiological culture, isolation and identification of species were performed by the University of Washington Clinical Microbiology Laboratory, according to standard clinical procedures. Briefly, samples submitted for “Lower Respiratory Culture for Cystic Fibrosis” (referred to hereafter as “CF sputum samples”) were mixed 1∶1 with 0.0648 M dithiotreitol (Sigma) and incubated for 5 minutes at room temperature, followed by vigorous vortexing for 1 minute. 50 µl aliquots of mucolysed sputa each were plated on sheep blood agar, MacConkey, chocolate, manitol salt, and cepacia agar culture plates. The remaining specimen was stored at −20°C until DNA extraction was performed.P. aeruginosa , 80%; B. cepacia , 14.11%, S. pyogenes, 5.65%; M. tuberculosis, 0.24%.DNA was extracted from isolated colonies or from the remaining volume of mucolysed sputa using a NucliSENS Easymag automated DNA extractor (BioMerieux). For CF sputa samples we included one extraction control per batch of 24 samples processed simultaneously. Abscess and lymph node biopsy material were purified with High Pure PCR Template Preparation Kit (Roche). Extracted DNAs were quantified by Qubit dsDNA HS kit (Life Technologies). For mixing studies, the relative contribution of 16S rRNA alleles from each organism was estimated from quantified input DNA, average genome size of sequenced reference strains, and average 16S rRNA locus copy number for the species. A mixture of 16S rRNA template from the following organisms was used: Bacterial genomic DNA isolated from isolates of clinical specimens was sequenced by the University of Washington Molecular Microbiology Laboratory using the Sanger method to establish 16S rRNA gene reference sequences or to attempt molecular diagnosis, where applicable.Three clinical specimens were excluded from the final analysis. CF13 and CF46 each generated too few de-noised bacterial sequence reads for meaningful analysis , likely secondary to poor balancing of the libraries. CF90 was also excluded, as we could not adequately confirm the identity of this specific specimen.The 16S rRNA gene contains nine variable regions (designated V1 to V9) Table S1. All PCR setup was performed in a laminar flow PCR workstation, and materials were UV irradiated prior to PCR setup. PCR to amplify 16S rRNA was carried out in two stages. Prior to the second stage, unincorporated primer was removed by DNA purification and additional cycles of PCR were performed using primers specific to the sequencing adaptor, amplifying only the molecules generated during initial PCR cycles. We found that this two-stage PCR strategy greatly reduces the amount of amplification from non-template controls.The sequences of PCR primers used for library preparation  are supplied in N_Barcode_357mI). To minimize amplification of contaminating bacterial DNA present in PCR reagents For the first round of PCR, a primer directed against the V1 flanking region (Primer P1) was used in conjunction with forward primers incorporating (from 5′ to 3′) Ion Torrent sequencing adaptor P1, a sample-specific “DNA molecular tag”, a 14-base semirandom sequence . The sequencing protocol was under development, particularly during the timeline of this project, and the details of the procedure had not been fully optimized for commercial release of 400 bp sequencing kits. For emulsion PCR, the protocols for the Ion PGM™ 200 Xpress™ Template Kit (Life Technologies) were modified to accommodate clonal amplification of the sequencing templates on to Ion Sphere Particles . The amount of ISPs and library molecules added to the emulsion was increased by 55%. A new polymerase and changes in salt conditions were also required for full extension of the longer template reads. PCR thermocycling conditions were modified as follows: one cycle of 95° for 6 minutes, 15 cycles of 95° for 30 seconds, 68° for 4 minutes, 30 cycles of 95° for 30 seconds, 68° for 6 minutes, then 10 cycles of 95° for 30 seconds, 68° for 20 minutes. Enrichment and quantification of template beads was performed according to manufacturer protocols.http://www.ncbi.nlm.nih.gov/sra), under study accession number SRP019805.400 bp semiconductor sequencing also required optimization in sequencing workflow and chemistries. A proprietary sequencing enzyme has been developed to increase both accuracy and read lengths, with concurrent optimization of flow order and nucleotide flow rates. Sequencing was performed on an Ion Torrent PGM (Life Technologies) using 800 flows (200 cycles), as opposed to the standard 520 flows. All sequencing was performed using 318 chips, with an approximate runtime of 7 hours per chip. Primary base calling was performed using Torrent Suite v3.0 software (Life Technologies), and sequences were exported in FastQ format. FastQ files were used for all subsequent analyses. Raw sequence reads for this project are available from the Sequence Read Archive (Sequencing reagents and protocols have subsequently been optimized and are available as Ion Torrent 400 bp sequencing kit (Life Technologies).We required that reads exceed 330 base pairs in length and contain one or fewer mismatches against a barcode sequence to pass initial filtering. Primer sites were identified in each read using the Smith-Waterman alignment algorithm (ssearch36) USEARCH v6 MUSCLE v3.5 De-noising of trimmed reads was accomplished by (1) performing a modified Figure S1). Parameters for de-noising were selected to maximize both the number of recovered reads and the pairwise identity of those reads compared to the appropriate reference sequence. We found that a clustering threshold of 98.5% or greater pairwise similarity combined with exclusion of clusters composed of fewer than 20 reads resulted in the most favorable combination of error rate and read recovery. For evaluation of non-template and extraction controls, clusters composed of 10 reads or greater were considered in order to further increase sensitivity.Parameters for de-noising were chosen empirically by calculating error rates as described below using sequences generated from control specimens containing a mixture of reference organisms with known 16S rRNA gene sequences  DeeNuRP (https://github.com/fhcrc/deenurp), and taxtastic (https://github.com/fhcrc/taxtastic). The second reference database was a collection of reference sequences for the purpose of classifying CF pathogens in comparison to culture (“CF pathogens”). To further minimize mis-annotation, we compared sequences in RDP-named to a collection of sequences spanning 16S rRNA V1–V2 or V1–V3, generated from clinical isolates identified in the Molecular Microbiology Laboratory at the University of Washington Medical Center. We used all sequences from clinical isolates representing genera containing any species designated as a CF pathogen. The CF pathogens database was then constructed by retaining any full-length sequence in RDP-named with both ≥99.5% identity (with at least 99% coverage) when aligned to a clinical sequence using BLAST, and with the same species-level taxonomic label.Candidate full-length 16S rRNA gene sequences (“RDP-full-length”) were downloaded from the Ribosomal Database Project  pplacer suite of tools  performs phylogenetic-based classification and population analysis by adding query sequences to a phylogenetic tree comprised of reference sequences DeeNuRP. Reference sequence selection for species of interest was performed by minimizing the average distance to the closest leaf (ADCL) of reads placed on a phylogenetic tree of candidate reference sequences as implemented in “guppy adcl” File S2) was assembled from sequences in RDP-named and was used for taxonomic assignment; the second  was assembled by comparing denoised reads to the RDP-full-length database. Multiple alignments of reference sequences were created using cmalignFastTreetaxtastic.The pplacer.We classified sequences using a combination of BLAST searches against curated databases of 16S rRNA sequences  and phylogenetic-based classification using Streptococcus mitis/oralis”) were constructed when reference sequences representing more then one species met these criteria. Consensus sequences with no qualifying matches were designated “no match”.We performed high-confidence species-level classification of denoised sequences on the basis of BLAST searches pplacer to perform phylogenetic placement of denoised reads onto the CF-named reference set described above. Multiple alignments of reads to reference alignments were created with cmalign. After placement, “guppy classify” was used to perform taxonomic assignment using default parameters. To remain consistent with conventions used in the clinical molecular microbiology lab for classifying closely related species, we modified the pplacer classification results as follows: any genus- or species-level names within family Enterobacteriaceae were renamed to “Enterobacteriaceae”; Pseudomonas hibiscicola was renamed to Stenotrophomonas maltophilia; any combination of Streptococcus mitis, S. oralis, S. pneumonia, or S. pseudopneumoniae was renamed to S. mitis/oralis/(pseudo)pneumoniae; any species belonging to the B. cepacia complex was renamed “Burkholderia cepacia complex”; and members of any combination of Achromobacter denitrificans, A. insolitus, or A. xylosoxidans was renamed to A. denitrificans/insolitus/xylosoxidans. In addition, reads classified by “guppy classify” as Pseudomonas aeruginosa group were renamed to P. aeruginosa on the basis of BLAST results for the same reads.To perform a more comprehensive taxonomic assignment of the CF sputum specimens, we used pplacer. To assess the stability of clades comprised by groups of specimens, we performed 100 bootstrap replicates; resulting trees were summarized using the script sumtrees.py as provided in DendroPy v3.3.1 Figure S2. Seven samples  were not assigned to any group because their composition was markedly divergent from other samples in the closest clade. For example, sample CF5, comprised primarily of reads classified as Acinetobacter spp., was clearly an outlier from Group II (dominated by Pseudomonas aeruginosa) and was therefore not included in that group. Each of the branches corresponding to these unassigned samples  was present in 100% of bootstrap replicates, consistent with strong support for their divergence from adjacent clades.CF specimens were grouped on the basis of the distribution and read mass (the cumulative number of reads contributing to clusters) and were placed onto the CF-unnamed reference tree by “squash” clustering To efficiently generate sequencing libraries we concatenated sequencing adaptors with PCR primers for amplification of a 16S rRNA sequencing target (variable regions V1–V2), which is sequenced clinically to achieve species-level classification of bacterial pathogens. Forward primers contained a 10-base “barcode” sequence to uniquely label products originating from a specific sample, allowing specimen multiplexing within the same sequencing run.The length of the target (∼360 bp) and artificial flanking sequences (∼30 bp) exceeded current capabilities of semiconductor sequencing, so it was necessary to develop new protocols to extend the read length. Emulsion PCR, used to prepare template molecules for sequencing by clonally amplifying them on beads, incorporated modified conditions for microdroplet formation and a new PCR enzyme was employed. Sequencing itself was performed using a novel DNA polymerase selected to maximize sequence quality while most efficiently synthesizing longer templates. Flow rates on the sequencing instrument were additionally optimized to improve accuracy and performance for longer template molecules.Libraries were pooled from an average of 16 samples each, and were sequenced on a single Ion Torrent PGM using 318 chips. An average of 3,374,183 reads  were obtained per chip. Full-length sequence reads comprised ∼18% of all reads  and compared the reads to corresponding Sanger sequences. The average per-read error rate varied per organism  that reduced the overall error frequency to an average of 0.633% per base per sequence  that were identified as B. cepacia. This finding suggests a low, but detectable, degree of cross-contamination. The cluster size cutoff of 20 reads, found optimal for de-noising, also excluded clusters containing reads attributable to this low level of cross-contamination.A related issue pertains to the possibility of cross-contamination between specimens. In addition to the low-level of contamination of presumed environmental origin, we amplified sequences from the non-template and extraction control specimens originating from organisms present in high concentration in CF sputa samples (1 to 13 raw reads), consistent with low-level cross contamination from clinical specimens. One specimen (CF38) contained a high concentration of To assess how effectively deep sequencing recovers low-prevalence bacterial species in a complex sample, we sequenced technical replicates of a mixture of purified DNA containing each of the four reference sequences. The estimated relative abundance of each template ranged from 0.25% to 80%. We explored the extent to which detecting minority species is limited by read depth by considering sensitivity given differing numbers of reads randomly subsampled prior to de-noising Figure 2Mycobacterium tuberculosis, which accounted for only 0.25% of 16S rRNA template in the original mixture, in all replicates. The objective of this experiment was primarily to demonstrate sensitivity and technical reproducibility, and we only have estimates of the relative proportion of each of the organisms represented in the mixture. Therefore, we cannot define the precision with which the relative abundance of each organism is reflected by read counts. Bias in the relative amplification efficiency of 16S rRNA from heterogeneous samples is a recognized artifact M. tuberculosis was consistently detected in simulated down-sampling of experimental data to as few as 5,000 reads, suggesting that depth of sequencing may provide some buffer against failing to detect minority species due to amplification and sampling bias should they exist.The relative representation of organisms was consistent between de-noised and unprocessed reads, across different library preparations of the same control specimen, across the concentrations of initial template DNA used, and among randomly selected subsets of reads of varying sizes. We detected sequences from Dataset S1). We focused on specimens that could not be adequately characterized by conventional techniques. To facilitate a direct comparison among methods, we used a BLAST-based classification, requiring at least 99% identity with a reference sequence to assign a classification. Although this conservative approach left a significant fraction of reads in some specimens unclassified at the species level (Table S3), it is consistent with criteria used in the clinical laboratory for classification by Sanger sequencing and provides a similar level of confidence in assigned classifications.One aim of this study was to assess the performance of deep sequencing relative to existing clinical microbiology techniques. To this end, we deeply sequenced challenging clinical samples and compared our results to those obtained using culture and Sanger sequencing of bulk PCR products (Dataset S1). Organisms identified were typical of human oral microbiota, including Streptococcus intermedius, Porphyromonas endodontalis, Prevotella oris, and Peptostreptococcus stomatis, which have been implicated as relevant organisms in brain abscess formation We first sequenced four brain abscess aspirates submitted for conventional molecular characterization by bulk 16S rRNA sequencing. Brain abscesses contain mostly non-viable organisms and therefore frequently fail identification by culture-based techniques Veillonella species based on the interpretation of a mixed-appearing, but still interpretable, electropherogram. Deep sequencing confirmed the presence of Veillonella species, but identified 16 additional bacterial species not detected by Sanger sequencing, presumably because they were detectable only as minor components of the mixed-appearing background. These findings indicate that even samples that are interpretable by Sanger sequencing may harbor a diverse, and otherwise unrecognized, bacterial population.We then sequenced a lymph node biopsy for which molecular characterization suggested a P. aeruginosa and members of the B. cepacia complex) or for identification of causative organisms during acute respiratory exacerbations. Samples were obtained without selection for patient characteristics or clinical indication for culture, and therefore represent a comprehensive sampling of patient samples during this period. These specimens were submitted with an order for “Lower Respiratory Culture for Cystic Fibrosis.” Because these specimens were otherwise de-identified, we cannot confirm the diagnosis of CF, and it is possible that some represent patients with other conditions. In parallel, our CLIA-certified clinical microbiology laboratory performed diagnostic sputum culture according to standard practices, and we performed deep sequencing of DNA purified from the remaining specimen (Dataset S1).Next, we examined sputum samples from cystic fibrosis (CF) patients, whose airways become chronically colonized by a complex mixture of phenotypically variable microbiota Stenotrophomonas maltophilia, Streptococcus agalactiae, Haemophilus influenzae, and Pseudomonas aeruginosa were detected more frequently by deep sequencing than by culture-based methods. Considering results for this set in aggregate, deep sequencing identified specific CF-relevant pathogens with greater frequency than culture . Conversely, in 22 cultured organisms (distributed across 17 of the 60 samples) were not reported by deep sequencing, with the most frequent example being S. aureus, which was detected by culture alone in 8 separate instances. Six of these missed organisms were recovered in de-noised clusters of less than 20 reads or identifiable using BLAST searches of the raw data (prior to de-noising), suggesting that loss of reads during de-noising at least partially accounts for these failures. Greater sequence read depth would presumably have resulted in detection of the missed organisms in these cases. For the remaining specimens, we found no correlation between failure rate and the relative abundance of the missed organisms based on culture (not shown). We also noted inconsistent mucolysis of unusually thick sputa in several samples, which may have resulted in non-homogenous sample aliquots separately being subjected to culture and DNA extraction.We first compared the ability of culture and deep sequencing to identify a targeted panel of CF pathogens of clinical interest, and whose presence in CF patient specimens is routinely evaluated by the clinical laboratory Table 2.pplacerCorynebacterium pseudodiphtheriticumTo more fully characterize the bacteria present in CF specimens, and to overcome limitations of a purely identity-based classification approach, we used the , Figure S2). Of the 66 sputum samples , 59 could be assigned to one of five major groups, reflecting broad similarities in microbial composition not apparent from culture results , including a Pseudomonas–dominant group (II), a Staphylococcus and Streptococcus-dominant group (IV), and three distinct, but more heterogeneous groups, composed mostly of Streptococcus and Prevotella (I), Streptococcus and Pseudomonas (III), or Pseudomonas with Prevotella and Streptococcus (V).We compared the bacterial communities among CF samples using “squash” clustering Next-generation sequencing technologies have gained increasing attention in the field of clinical microbiology Building upon metagenomic research strategies and existing clinical methods for molecular bacterial characterization, we developed an approach for classifying the species present in clinical samples containing complex bacterial communities using deep sequencing. Semiconductor next-generation sequencing (Ion Torrent) offers rapid chemistries that make it amenable for adaptation as a clinical diagnostic tool, so was selected as the sequencing platform in this study. Subsequent improvements to the workflow with commercial release of Ion Torrent 400 bp sequencing kits have made the assay described theoretically compatible with same day turnaround times , potentially allowing for results to be returned faster than can be achieved by culture. In conjunction, multiplexing specimens through DNA barcoding allows significantly reduced per-sample costs Dataset S1), providing the level of accuracy necessary for clinical diagnosis. It should be possible to further decrease errors among de-noised reads by selecting only clusters containing large numbers of reads, but at the expense of decreasing sensitivity secondary to excluding rare sequences.We found that sequencing errors for the assay (integrating library construction and sequencing) are largely secondary to artifacts involving indels, a well-known limitation of semiconductor sequencing, and are similar to published error rates for Ion Torrent PCR-mediated deep sequencing library preparation allows highly-purified libraries to be quickly generated from trace quantities of bacterial DNA, in contrast to shotgun sequencing approaches which are less efficient and nonspecifically produce sequence data from the human host As an applied proof-of-principle we have explored the composition of challenging clinical specimens, demonstrating key advantages of molecular microbiology diagnosis by next-generation sequencing. Deep sequencing proved most useful in providing actionable information about the microbial composition of brain abscess material, whereas both Sanger sequencing and standard culture failed to provide a result. Similarly, deep sequencing cataloged a number of bacterial species from a biopsy which were not resolvable by Sanger sequencing, and which was clinically reported as infection with a single organism.Dataset S1). As expected S. aureus was the organism most frequently missed by deep sequencing, consistent with earlier reports using quantitative real-time PCR In addition to materials where bacteria cannot be effectively cultured or sequenced by the Sanger method, we also explored the utility of deep sequencing using a collection of CF sputa that were simultaneously characterized using standard clinical practice microbiology culture , CF pathogens that are not resolved by routine clinical culture Dataset S1). Thus, the true number of CF pathogens diagnosable by deep sequencing is greater than reported with respect to the limited panel of organisms surveyed by culture.Further optimization will be required before deep sequencing is suitable as a stand-alone diagnostic for CF sputa. Regardless, even currently deep sequencing detected specific CF pathogens from a greater number of patient specimens than culture, indicating utility as an adjunct identification technique. Moreover, members of the , Figure S4), which are not apparent based on conventional culture results. This finding suggests that a diverse CF patient population can be binned into a limited number of categories given the makeup of their respiratory microbiota. Two of the groups (II and IV) have relatively low diversity and are dominated by combinations of Staphylococcus, Streptococcus, and Pseudomonas; all well-described colonizers of the airway of CF patients. Groups I, III, and V are more diverse. Groups I and V each contain a substantial fraction of obligate anaerobes including Prevotella, Veillonella, and Porphyromonas species. Anaerobic organisms have been noted in CF sputa in a number of studies It may prove more informative to evaluate the overall microbial population in a patient's airway rather than to screen for specific pathogens There are several additional considerations to the use of 16S rRNA deep sequencing in the clinical laboratory. First, although de-noising strategies have proven valuable, their use prevents discrimination among closely related strains. Because de-noising functions by clustering similar reads that are assumed to derive from the same template molecule, sufficiently similar sequences may be integrated into a single consensus. Therefore, although our approach can accurately and sensitively “rule in” bacteria whose sequences closely match those in a database of known 16S rRNA genes, it currently does not allow certain bacterial species to be “ruled out” from clinical specimens in cases where a closely related species is also detected. We expect that future improvements in PCR enzyme cocktails, sequencing chemistries, and primary base-calling algorithms will reduce rates of raw sequencing error on this platform, decreasing reliance on de-noising algorithms and improving the resolution of the assay. More sophisticated de-noising algorithms incorporating error models specific to semiconductor sequencing may also prove beneficial Despite these caveats, deep sequencing demonstrates the potential for immediate utility in several clinical applications exemplified by this study, namely, characterizing mixed infections from specimens containing non-viable or unculturable organisms, such as brain abscesses or fixed tissues, and detecting specific bacterial pathogens from complex specimens when a defined list of species are of interest, such as CF sputa Dataset S1Classification results for clinical specimens.(XLSX)Click here for additional data file.File S1De-noised sequences.(TGZ)Click here for additional data file.File S2CF-named reference package.(BZ2)Click here for additional data file.File S3CF-unnamed reference package.(BZ2)Click here for additional data file.Figure S1Overall error rates for different de-noising parameters.(PDF)Click here for additional data file.Figure S2Squash clustering of CF sputa microbiota. Bootstrap support values are indicated along corresponding nodes.(PDF)Click here for additional data file.Figure S3Genus-level classification performed by analysis of de-noised deep sequencing reads using pplacer. The relative number of each species (by read count) is represented by the height of corresponding bars. Phylogenetic “squash” clustering of specimens from deep sequence data is represented as a cladogram, with specimens colored as in (PDF)Click here for additional data file.Figure S4Consensus microbiota profile of phylogenetic groups at the genus-level, averaged from all members of the group.(PDF)Click here for additional data file.Table S1Primer Sequences.(XLSX)Click here for additional data file.Table S2Composition of sequencing libraries.(XLSX)Click here for additional data file.Table S3Putative genus-level classification of consensus sequences from brain abscesses and lymph node biopsy that were unassigned at the species-level.(XLSX)Click here for additional data file.