Using Taqman assays with probes targeting specific exon-exon junctions, we here demonstrate that ddPCR reproducibly quantified cDNA fragments with and without exon 51 of the DMD gene over a 4-log dynamic range. In a comparison of conventional nested PCR, qPCR and ddPCR using cDNA constructs with and without exon 51 mixed in different molar ratios using, ddPCR quantification came closest to the expected outcome over the full range of ratios (0–100%), while qPCR and in particular nested PCR overestimated the relative percentage of the construct lacking exon 51. Highest accuracy was similarly obtained with ddPCR in DMD patient-derived muscle cells treated with an AON inducing exon 51 skipping. We therefore recommend implementation of ddPCR for quantification of exon skip efficiencies of AONs in (pre)clinical development for DMD.Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required. With the recent introduction of 3 DMD gene located on chromosome Xp21, which are mostly intragenic deletions (72%) resulting in loss of one or more exons and disruption of the dystrophin open reading frame . The LinRegPCR method, which uses linear regression analysis to determine the PCR efficiency per sample based on the slope of the exponential phase of the amplification curve, required the raw fluorescent data (not baseline corrected) to be imported in LinRegPCR software version 2014.6. The mean PCR efficiency per amplicon (E) was used to calculate a starting concentration per sample (N0), expressed in arbitrary fluorescence units using the equation N0 = Nt/ECq where Nt is the fluorescence threshold and Cq is the cycle number per sample [0 skipped/(N0 skipped + N0 non-skipped)*100].Real time quantitative PCR (qPCR) was performed in triplicate on 2 μl of cDNA in a 10 μl reaction volume containing 0.5 μl of the relevant Taqman assay, 5 μl of Gene expression master mix  and 3 μl of DNase/RNase-free Hl method  and the l method . The Pfar sample . Data wa2O. In each ddPCR run multiple technical replicates were run in parallel and a no template control (NTC) was included as negative control. Each 20 μl PCR reaction was loaded in an 8-channel disposable droplet generation cartridge. Seventy μl of droplet generation oil  was loaded in adjacent oil wells on the cartridge and the microfluidic chip was placed in the QX200 droplet generator (BioRad). During this process the sample mix was partitioned into ~20,000 nano droplets of equal size, randomly distributing target molecules into droplets. The resulting water-in-oil emulsion was pipetted from the outlet well to a semi-skirted 96 well PCR plate . After transfer of all samples, the plate was sealed with foil and amplified on a T100 thermal cycler (BioRad) using the following protocol: 95°C for 10 min, 40 cycles of 95°C for 15 sec and 60°C for 1 min, and 98°C for 10 min to inactivate enzymatic nuclease activity. Plates containing the amplified droplets were loaded into the QX200 droplet reader (BioRad), which aspirates droplets from the 96-well plate, one well at a time, and streams them in single file past a detector for FAM and VIC/HEX fluorescent dyes. On average data from 12,000–18,000 droplets per sample was used in subsequent calculations.Digital droplet PCR (ddPCR) was performed on 2 μl of cDNA in a 20 μl reaction volume containing 1 μl of the relevant Taqman assay, 10 μl of ddPCR Super mix for probes  and 7 μl of DNase/RNase-free HDiscrimination between droplets without target (negatives) and droplets with target (positives) was achieved by applying a fluorescence amplitude threshold. For both the DMD ex47-52 and DMD ex51-52 Taqman assays, a threshold of 4,000–5,000 relative fluorescent units (RFU) was applied, but was reset manually to a different value if this gave a better discrimination between the positive and negative droplet population. Digital PCR runs were considered technically successful if the number of accepted droplets per individual sample replicate was >10,000. To assess variability between technical replicates the coefficient of variation (%CV = (stdev/avg)*100) was calculated. The concentration (in copies/μl sample mix) of the skipped assay and non-skipped assay was used to calculate the percentage exon skip [copies/μl skipped/ (copies/μl skipped + copies/μl non-skipped)*100].DMD cDNA fragments with (DMD ex51-52.2) or without exon 51 (DMD ex47-52) in a DMD mutational background of an exon 48–50 deletion (Δ48–50), which occurs in ~7% of the DMD deletions [Taqman assays were designed to specifically detect eletions . The speTo demonstrate the reproducibility of ddPCR quantification over a wide dynamic range of template concentrations, 10-fold serial dilutions of dystrophin cDNA constructs with and without exon 51 were generated and used as template in ddPCR analysis using the DMD ex47-52 and DMD ex51-52.2 assays . Using 4To determine the precision of the DMD ex51-52.2 and DMD ex47-52 ddPCR assays, different concentrations of the dystrophin cDNA constructs with and without exon 51 were measured in 8 replicates per concentration . The obsDMD cDNA construct template mixtures using the DMD ex47-52 and the DMD ex51-52.2 assays yielded an outcome highly identical to the expected percentages based on molar ratio of the mixed constructs. This was the case over the entire range of template mixtures, demonstrating the quantification accuracy of ddPCR  transfected with ex51 AON . Total RAdditional Taqman assays were developed for four other deletions amenable to exon 51 skipping: Δ45–50, Δ49–50, Δ50, and Δ52 . The speTargeted exon skipping using antisense oligonucleotides (AONs) to correct reading frame disrupting deletions is a therapeutic approach that is in late stage clinical development for DMD. Since this is a mutation-dependent approach, several different AONs are required to target the majority of the DMD mutations occurring in the mutational hotspot region between exons 45 and 55. Drisapersen and eteplirsen are designed for DMD patients whose mutation is amenable to exon 51 skipping, constituting approximately 13% of the DMD population. While the primary endpoint to assess efficacy in clinical trials is the six minute walk test , dystropSince the primary anticipated mechanism of action of AONs for DMD is induction of exon skipping, measuring exon skip levels in muscle biopsies from treated patients may serve as a supportive molecular marker. However, as a majority of DMD patients have a low level of spontaneous skipping of exons flanking their deletions, accurate quantification of exon skipping levels in pre- versus post-treatment biopsies is essential. Unfortunately, the conventional nested PCR method so far widely used in the exon skipping field to measure exon skip levels is qualitative or at best semi-quantitative and has resulted in a systematic and widespread overestimation of exon skip levels.Recently, due to technological advances improved PCR-based methodologies have become available. Here we show that third generation ddPCR is an extremely sensitive, reproducible and reliable method for absolute quantification of exon skip levels in (pre)clinical samples. We have demonstrated that ddPCR can reliably quantify exon skip levels over a wide 4-log dynamic range of template concentrations and that the outcome is very robust and not influenced by differences in PCR efficiency. It is worth noting that ddPCR is not more time consuming than conventional nested PCR followed by lab-on-chip analysis. Furthermore, ddPCR is highly sensitive and can detect a very low level of spontaneous exon 51 skipping occurring at baseline in a DMD muscle cell culture. To allow applicability of this methodology for muscles biopsy analysis in clinical studies, we have developed Taqman assays for 5 different DMD deletions amenable to exon 51 skipping. Together these mutations represent the majority of the total number of DMD deletions correctable by exon 51 skipping. We have demonstrated the specificity of all these assays on cDNA constructs that reflect the exon content of the different mutations with and without exon 51 skipping. In addition, we have also quantified exon 51 skip levels in vitro in patient derived muscle cell cultures transfected with ex51 AON. When comparing the ability of different PCR-based methods to accurately quantify different molar ratios of cDNA constructs with and without exon 51, ddPCR came closest to the theoretical calculated outcome over the full range of ratios (0–100%). In contrast, qPCR overestimated the exon skip levels in particular when the LinRegPCR method was applied to correct for differences in PCR efficiency between the assays for the cDNA constructs with and without exon 51. This can largely be explained by the difference in mean efficiencies between the assays calculated by the LinRegPCR software. The mean efficiency of the DMD ex47-52 assay was calculated to be approximately 11% lower than that of the DMD ex51-52.2 assay, resulting in an overestimation of the exon 51 lacking/containing construct ratio. This overestimation of exon skip was even more pronounced using nested PCR, and increased with the total number of PCR cycles, probably due to amplification bias towards the shorter amplicon.Although ddPCR offers a precise, reproducible and absolute quantification of exon skip levels, the measurement is similarly impacted by several upstream factors that may influence outcome and results should always be interpreted carefully taking technical and biological limitations into account. For example, muscle biopsies obtained from DMD patients often have a lower muscle fiber content and more fibrotic or fatty tissue than in healthy donor biopsies, resulting in lower levels of non-skipped dystrophin expression and potentially hampering reliable quantification of exon skip levels. In addition, it is not always feasible to isolate high quality RNA from patient biopsies, which may lead to increased variation. Furthermore, efficiency of the reverse transcription step to convert RNA to cDNA may introduce variation in ddPCR measurements. Since different cDNA priming methods are known to generate different outcomes , use of DMD exon specific primers, probes and assays is that the complexity of the dystrophin transcriptome can never be entirely covered using Taqman assays or PCR amplicons that specifically detect or amplify a single exon-exon junction. The dystrophin pre-mRNA is subject to extensive alternative splicing [DMD transcript levels has been reported to occur in skeletal muscles of mdx mice and in patients with Becker muscular dystrophy, with reduced transcript levels toward the 3′ end [Another consideration to take into account when monitoring exon skip levels using splicing , which ge 3′ end . This 5′Despite these limitations that more generally apply to working with patient biopsies, the actual quantification of exon skip levels in a given cDNA sample using ddPCR has a high precision, sensitivity and reproducibility and importantly does not overestimate exon skip. Since accurate detection of increased exon skipping levels in post-treatment biopsies would be supportive for an AON drug effect, we thus recommend ddPCR technology for quantification of exon skip efficiencies in the various stages of (pre)clinical development of AONs for DMD.S1 FigSpecificity of the Taqman assays representative for the exon 48–50 deletion transcripts in qPCR analysis and subsequent gel electrophoresis. (A) The specificity of both DMD ex51-52.2 and DMD ex47-52 Taqman assays was confirmed by qPCR analysis using cDNA from a Δ48–50 cell line. The DMD ex47-52 assay detected the skipped product in treated cells, whereas nothing was detected in NT samples. (B) Gel analysis of qPCR products for both DMD ex47-52 and DMD ex51-52.2 Taqman assays. Primers for the DMD ex47-52 Taqman assay also amplify the transcript in which exon 51 is still included, resulting in two bands differing in size by approximately 200 bp, which corresponds to the size of exon 51 (233bp). However, specificity for the skipped product is provided by the Taqman probe directed against the exon 47–52 junction, which only generates a fluorescent signal for the smaller product lacking exon 51(C). Gel analysis of qPCR products for Taqman assays that detect skipped transcripts  and non-skipped transcripts (DMD ex51-53) in ex51 AON treated patient derived myotubes with different mutational backgrounds. DMD ex44-52.3, DMD ex48-52, DMD ex49-52.2 & DMD ex50-53 assays all show two products separated by ~233 bp (the size of exon 51): the transcript without exon 51 (lower band) and the transcript containing exon 51 (upper band). Specificity for the skipped product is provided by the Taqman probe which which only generates a fluorescent signal for the smaller product lacking exon 51.(TIF)Click here for additional data file.S2 FigSpecificity and efficiency of the Taqman assays representative for the exon 48–50 deletion transcripts in qPCR analysis. (A) The specificity of both DMD ex51-52.2 and DMD ex47-52 Taqman assays was confirmed by qPCR analysis using dystrophin cDNA constructs representative for Δ48–50 transcript fragments with or without exon 51. (B) 10-fold serial dilution of a 50–50 mixture of the dystrophin cDNA construct templates representative for Δ48–50 transcript fragments with or without exon 51.(TIF)Click here for additional data file.S3 FigTaqman assays were designed to detect exon 51 skipping in samples of DMD patients with different relevant deletions using the guidelines of the Primer express software (Applied Biosystems). For the specific detection of transcripts containing exon 51, the forward primer was located in exon 51 and the reverse primer in exon 52, while the Taqman probe spans the exon-junction. For the samples with an exon 52 deletion, an alternative assay was designed to detect the transcript containing exon 51.(TIF)Click here for additional data file.S1 TableDystrophin cDNA constructs, representative for the transcripts arising from several exon 51 skip amenable deletions and the resulting transcript fragment following exon 51 skipping, were synthesized by Life Technologies. All cDNA constructs had a length of 1200bp and were verified by sequencing. Dark shaded exons were included entirely and light shaded exons were partially included.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Obtaining high-quality samples from wild animals is a major obstacle for genomic studies of many taxa, particularly at the population level, as collection methods for such samples are typically invasive. DNA from feces is easy to obtain noninvasively, but is dominated by bacterial and other non-host DNA. The high proportion of non-host DNA drastically reduces the efficiency of high-throughput sequencing for host animal genomics. To address this issue, we developed an inexpensive capture method for enriching host DNA from noninvasive fecal samples. Our method exploits natural differences in CpG-methylation density between vertebrate and bacterial genomes to preferentially bind and isolate host DNA from majority-bacterial samples. We demonstrate that the enrichment is robust, efficient, and compatible with downstream library preparation methods useful for population studies . Compared to other enrichment strategies, our method is quick and inexpensive, adding only a negligible cost to sample preparation. In combination with downstream methods such as RADseq, our approach allows for cost-effective and customizable genomic-scale genotyping that was previously feasible in practice only with invasive samples. Because feces are widely available and convenient to collect, our method empowers researchers to explore genomic-scale population-level questions in organisms for which invasive sampling is challenging or undesirable. The past decade has witnessed a rapid transformation of biological studies with the continuing development and adoption of massively parallel sequencing technology. This sequencing revolution, however, has thus far had a relatively muted impact on studies of wild nonmodel organisms due largely to the difficulty of obtaining high-quality samples. This problem is particularly salient for endangered animals, cryptic animals, or animals for which it is otherwise difficult, undesirable, or unethical to obtain samples invasively.1. Of these, feces may be the most readily available in many taxa2. Indeed, since PCR amplification of DNA from feces was first demonstrated in the 1990s3, noninvasive genetic studies from feces have revolutionized our understanding of the evolution, population structure, phylogeography, and behavior of nonmodel organisms. PCR amplification, however, is effective only for short sequences of DNA. The ability to generate cost-effective genomic-scale data of animals from feces using massively parallel sequencing would therefore constitute an important methodological advance towards bringing a greater number of wild organism studies into the genomic age.Field researchers working with wild animals have explored several noninvasive sample types for DNA analysis including feces, hair, urine, saliva, feathers, skin, and nails1, particularly with long amplicons. Finally, endogenous (host) DNA in feces constitutes a very low proportion, typically less than 5%6, of total fecal DNA. Instead, fecal DNA contains a preponderance of DNA from exogenous (non-host) sources such as gut microbes, digesta, intestinal parasites, and environmental organisms. Gut bacteria pose a particular challenge as they account for the highest proportion of DNA in feces5.Feces presents significant challenges for genetic analysis. DNA in feces is often fragmented and low in quantity. Fecal DNA extractions are further characterized by a frequent presence of co-extracted PCR inhibitors, sometimes complicating PCR detection of genotypesBecause of the high representation of exogenous DNA in feces, shotgun sequencing of fecal DNA would yield only a small proportion of reads matching the host genome. For genomic studies of host organisms, particularly those targeting populations, this represents a crippling obstacle in the presence of typical financial constraints. Without an effective enrichment procedure, sequencing of fecal DNA would be less efficient than that of invasively obtained “high-quality” DNA by at least one order of magnitude regardless of improvements in sequencing throughput or cost.6 have thus far employed targeted sequence capture methodologies. Sequence capture, like PCR, enriches DNA based on sequence specificity but unlike traditional PCR can work at any scale from a single locus7 to a whole genome9. This method involves hybridizing DNA or RNA “baits,” either affixed to an array11 or to magnetic beads in solution12, to a mixture of target and nontarget sequences, thereby capturing targeted DNA from the mixture. Sequence capture has been used for instance to enrich human exomes13, reduced-representation genomes16, host DNA from ancient or museum specimens19, and pathogen genomes from human clinical samples8. While the cost of custom oligonucleotide bait synthesis remains high, methods for transcribing custom baits from existing DNA templates9 have driven costs significantly down, increasing sequence capture’s appeal.Attempts to enrich host DNA from feces for genomic analysiset al.5 first successfully enriched host DNA from feces at the genomic scale. Using a modified sequence capture employing custom-synthesized baits, they were able to highly enrich 1.5 megabases of chromosome 21, the X chromosome, and the mitochondrial genome from fecal samples of 6 captive chimpanzees. Their protocol, however, remains prohibitively expensive for population-level analysis due to the high cost of bait synthesis. More recently, Snyder-Mackler et al.6 performed whole-genome capture on fecal DNA, using RNA baits transcribed in vitro from high quality baboon samples to enrich host genomes from 62 wild baboons. Resulting libraries were sequenced to low coverage (mean 0.49×), but nevertheless provided sufficient information for reconstructing pedigree relationships.Perry 6. Because both RNA baits and the gDNA used to transcribe them are eventually depleted, the composition of RNA baits varies between bait sets, potentially impeding comparison of samples sequenced using different RNA baits and gDNA templates. Trans genomic captures (i.e. capturing DNA using baits from a different species) may complicate enrichment and introduce at least some capture biases20, which will be a particular impediment for genomic studies for which high-quality DNA from related taxa is not accessible. Sequence capture may also introduce biases toward the capture of low-complexity, highly repetitive genomic regions, as well as an excess of fragments from the mitochondrial genome21.Despite these methodological advances, targeted sequence capture has distinct drawbacks. To avoid the high cost of bait synthesis, RNA baits must first be transcribed from high-quality genomic DNA that is consumed by the process, limiting its appeal when working with species for which high-quality DNA is difficult to obtain or in short supply. The processes of both bait generation and hybridization with fecal DNA are labor-intensive and time-consuming, with the hybridization including an incubation step that alone takes 1–3 days22, our method employs a bait protein created by genetically fusing the human methyl-CpG binding domain protein 2 (MBD2) to the Fc tail of human IgG1. The resulting MBD2-Fc protein is then bound by a paramagnetic Protein A immunoprecipitation bead to create a complex that selectively binds double-stranded DNA with 5-methyl CpG dinucleotides. Because vertebrate DNA contains a high frequency of methylated CpGs24 while bacterial DNA does not26, this MBD bait complex selectively binds host DNA  exist.We have developed a method that makes noninvasive population genomics economically and practically feasible for the first time, by exploiting natural, evolutionarily ancient differences in CpG-methylation densities between vertebrate and bacterial genomes to enrich the host genome from feces. This method, which we call FecalSeq, uses methyl-CpG-binding domain (MBD) proteins to selectively bind and isolate DNA with high CpG-methylation density. Modified after techniques to enrich the microbiome from vertebrate samples22.Our enrichment approach captures eukaryotic DNA using a methylated CpG binding domain protein fused to the Fc fragment of human IgG (MBD2-Fc) to selectively target sequences with high CpG methylation densityTo evaluate our approach, we enriched DNA extractions from the feces of 6 captive and 46 wild baboons, which we then used to prepare and sequence ddRADseq libraries. We also prepared ddRADseq libraries from blood-derived genomic DNA of all six captive baboons to facilitate controlled  comparisons of blood and fecal libraries. All libraries were sequenced using Illumina sequencing.Quantitative PCR estimates of starting host DNA proportions in fecal DNA extracts ranged widely, but were substantially lower in samples obtained from the wild . Endogenous DNA proportions on average increased 13-fold as estimated via comparison of pre-enrichment host proportion (from qPCR) and post-enrichment proportion of reads mapped .Based on two pilot libraries constructed from MBD-enriched fecal DNA, we found that there was large variation in the proportion of reads mapping to the baboon reference genome , we created and sequenced a third library from MBD-enriched fecal DNA. After noting substantial improvements in enrichment, we finally sequenced a fourth library with MBD-enriched fecal DNA from 40 wild baboons.Despite having similar or even lower starting host DNA proportions, read mapping proportions in the third library were substantially higher than the prior two . Following enrichment, however, host DNA proportions were nonetheless higher than our pilot libraries  computed from same-individual RADtags exhibited in some cases higher values for feces-derived samples , indicating low allelic dropout attributable to the MBD enrichment. For this test, we also controlled for variation in sequencing coverage as described above.Dropout of entire RADtags is easily detectable given a reference genome or sufficient samples for comparison; dropout of a single allele at heterozygous sites is a more insidious potential bias. Allelic dropout due to MBD enrichment would result in a decrease in heterozygosity in MBD-enriched fecal libraries. Inbreeding coefficients  using PLINKace Fig. . The res30.Stringent filtration of SNP sets, as would be implemented in a standard population genetic study, reduced the apparent biases attributable to fecal enrichment, measured both as total SNPs with a significant association with sample type  as well as total SNPs with significant missingness assessed via a chi-square test . Though more work is needed to quantify more exactly the extent and causal factors that lead to missingness, many population genetic analyses are robust to the low level of dropout our analyses reveal in addition to that which is inherent in the RADseq family of techniquesOur methylation-based capture method achieves substantial enrichment of host DNA from fecal samples. Using our revised protocol developed through experimentation, we produced a mean 195-fold enrichment on our final library consisting entirely of fecal DNA obtained noninvasively under remote field conditions, with most samples nearly a decade old. A mean 28.8% of reads mapped to the baboon genome, despite starting with only a mean 0.34% of host DNA. Using fecal and blood DNA obtained from captive animals, we further demonstrate that feces-derived genotyping data following our method are concordant with corresponding data obtained from blood.1, and are particularly useful for population-level studies or studies of endangered or elusive species for which obtaining high-quality samples is difficult or undesirable. By exploiting methylation differences rather than sequence differences between host and bacterial DNA, FecalSeq is an enrichment strategy that requires neither prior genome sequence knowledge nor the use of high-quality DNA for preparation of capture baits. This results in enrichment which is both inexpensive and replicable. The enrichment procedure is also relatively rapid and uncomplicated. Using a 96-well plate, we performed two sequential rounds of enrichment on all forty samples in our final library within a day .Feces are among the most readily accessible sources of information on wild animals31 which add a very low cost per sample (about $0.10 per sample).Compared to comparable experiments using high-quality DNA samples such as blood, our enrichment method introduces extremely low added costs. After excluding shared costs such as DNA extraction, library preparation, and sequencing, major costs associated with our method are qPCR reagents for initial quality assessment of fecal DNA samples and enrichment reagents for capturing the host genome. qPCR reagents cost about $0.60 USD per reaction (or $1.20 per sample assuming samples are run in duplicate). For our enrichment protocol, the amount of reagents used will vary based on the starting proportion of host DNA in the sample . Assuming fecal DNA samples on average contain 2.5% host DNA, a single enrichment kit will support a total of 240 enrichment reactions at $0.70 per sample. Based on our experience, most fecal DNA samples contain less than 2.5% host DNA and will therefore require less reagents, further lowering the cost per sample. Following enrichment, we purified DNA using homemade SPRI beads6, which at present represents the only other published strategy for enriching fecal DNA. Snyder-Mackler et al.6 present the most cost-effective sequence-capture-based strategy to date at a cost of $60 per sample, or $29 per sample when using a multiplexed capture strategy on 10 samples at a time. Overall, our strategy therefore decreases the cost of enrichment by about one order of magnitude.Altogether, compared to high-quality DNA experiments, the marginal cost of our method is approximately an additional $2 USD per sample factoring in duplicate qPCR reactions, enrichment, and SPRI bead cleanup. These costs represent a substantial decrease relative to enrichment methods based on oligonucleotide-based sequence capture27, there is substantial flexibility for researchers to optimize the number of samples and the fraction of the genome sequenced for particular research questions. This is not possible for libraries prepared using targeted sequence capture, which are therefore currently limited mainly to low-coverage analyses at the population level6. Transcription of sequence capture baits from reduced-representation libraries may potentially help address this problem16, but its efficacy for fecal DNA has yet to be demonstrated.Importantly, FecalSeq is to our knowledge the first genomic-scale fecal DNA enrichment method that is compatible with most downstream library preparation methods for massively parallel sequencing. Through our use of ddRADseq, we demonstrate that our method facilitates low-cost high-capacity genotyping of wild populations without introducing significant bias. Further, because ddRADseq is customizableAlthough ideally suited to taxa with a reference genome, genotyping via double-digest RADseq is possible for species that lack a closely related reference genome. We aligned our sequencing reads to the baboon reference genome for this study, but our approach is likely also applicable to species without a reference genome. A reference genome from a more phylogenetically distant species can be used, although reads from divergent regions will fail to map. If a nearby genome is not available, an additional pre-screening step would be necessary, in which exogenous reads are filtered out through comparison to the nearest available genome, before proceeding to clustering and variant identification as per normal reference-free ddRADseq methods. Although these caveats will be mitigated as more genomes become available, the proximity of study individuals to an available reference genome is an important consideration when deciding if ddRADseq methods, including ddRADseq following our enrichment method, are suitable to address the research goals.6. Future attention should therefore be directed towards fecal sample collection, storage, and extraction methods that maximize the selective recovery of host nuclear DNA32. While we demonstrated effective genotyping of samples with often very low starting proportions of host DNA (the vast majority < 0.5%), future studies may consider pre-screening extracted DNA samples using qPCR to select for samples with high starting proportions of host DNA.We robustly found that sequencing efficiency (percentage of reads assigned to target genome) of MBD-enriched fecal DNA libraries correlates strongly with starting proportions of host DNA, echoing findings using other capture methodsWe found that, for unknown reasons, starting host DNA concentration for fecal samples collected in captive conditions was higher than that of fecal samples collected from the wild, despite consistent sample collection and processing protocols. We speculate that these differences may be due to either the freshness of samples collected in captivity or to dietary differences between captivity and the wild. Because our wild fecal samples were collected under common biological field conditions , our study underscores the need to validate enrichment methods for wild animals using samples from the wild.Low starting proportions of host DNA present a challenge not only because they result in lower sequencing efficiency, but also because they correlate with low absolute quantities of DNA belonging to the host organism. In some cases, particularly in samples collected from wild animals under field conditions, starting proportions of host DNA were so low that only approximately 0.1 ng of target DNA was available in a 1 μg fecal DNA extract. Given the large genome sizes of baboons (approximately 3 Gb) and many other vertebrates, substantial allelic dropout is expected in these cases. Significantly, this challenge cannot be fully addressed by this or any other enrichment method and remains an important consideration for researchers working with feces. It can be minimized, however, by optimizing the enrichment procedures to maximize the recovery of target DNA present in a fecal DNA sample, as well as by increasing the total amount of starting fecal DNA.33. While this may be undesirable for studies requiring the matrilineally inherited marker, it also precludes the disproportionately high representation of mitochondrial DNA that is typical in libraries prepared using the targeted sequence capture approach21. FecalSeq may, however, co-enrich nuclear DNA from exogenous eukaryotes such as from plant or animal digesta. Care should therefore be taken to minimize the presence of exogenous eukaryotic tissues or cells, although the degree to which this is a problem in practice is currently unknown. As cell-wall-bound plant cells may be more likely to pass through the digestive tract intact, extraction methods that minimize lysis of cell walls should be preferred. We speculate that prey DNA from carnivorous animals may be more difficult to partition from host DNA. While our comparisons of inbreeding coefficients, unique alleles, and locus-by-locus statistical tests from matched blood and fecal samples revealed no significant effect of sample type and therefore enrichment, our small sample size limits our power to detect differences with small effect size. Subsequent studies with larger sample sizes may detect a significant effect of enrichment, but one which may be negligible or correctable for some applications.Because MBD enrichment partitions DNA based on CpG-methylation density, FecalSeq does not enrich hypomethylated host mitochondrial DNA35, noninvasive genetic studies have revolutionized our understanding of the evolution, ecology, and behavior of nonmodel organisms. By facilitating low-cost genomic-scale sequencing from feces, our method connects a community of field researchers with the benefits of massively parallel sequencing, ushering noninvasive organism studies into the genomic age.Since PCR amplification of DNA from feces was first achieved in the 1990sPapio) housed at the Southwest National Primate Research Center (SNPRC) at the Texas Biomedical Research Institute. The individuals were of either P. anubis or hybrid ancestry . Sedation and blood draws were performed under the supervision of a veterinarian and animals were returned immediately to their enclosures following recovery. Sample collection in Zambia was conducted with approval by the Animal Studies Committee of Washington University (assurance #A-3381–01) and following local laws and regulations in Zambia.Buffy coat extractions were performed using the QIAamp DNA Blood Mini Kit (Qiagen), following manufacturer’s instructions. Fecal extractions were performed using the QIAamp DNA Stool Mini Kit (Qiagen) following manufacturer’s instructions for optimizing host DNA yields. DNA concentration and yield were measured using a Qubit dsDNA BR Assay (Life Technologies). In some cases, multiple DNA extractions from the same individuals were necessary when DNA was depleted over the course of this study.MYCBP primers36 and evaluated against a standard curve constructed from the liver DNA of an individual baboon. Samples and standards were run in duplicate alongside positive and negative controls .We estimated the proportion of host DNA for each fecal DNA extraction using quantitative PCR (qPCR) by comparing estimates of host DNA concentration obtained by qPCR to estimates of total fecal DNA concentration obtained by Qubit. Amplification was conducted using universal mammalian 22.DNA was enriched using the NEBNext Microbiome DNA Enrichment Kit (New England Biolabs)n μl batch, we prebound 0.1 × n μl MBD2-Fc protein to n μl protein A magnetic beads by incubating the mixture with rotation for 10 min at room temperature. The bound MBD2-Fc magnetic beads were then collected by magnet and washed twice with 1 ml ice-cold 1x bind/wash buffer before being resuspended in n μl ice-cold 1x bind/wash buffer.MBD2-Fc-bound magnetic beads were prepared according to manufacturer instructions in batches ranging from 40 to 160 μl. For each 31, and quantified using a Qubit dsDNA HS Assay (Life Technologies).As a pilot experiment, we prepared two successive libraries, library A and library B, following manufacturer’s instructions for capturing methylated host DNA, with minor protocol modifications incorporated for the second pilot library (library B). Library A included MBD-enriched fecal DNA from 4 SNPRC baboons and 2 Luangwa Valley baboons, as well as blood DNA from the same SNPRC baboons. Library B included MBD-enriched fecal DNA from 4 SNPRC baboons (with two repeats from library A), 4 Kafue National Park baboons, and 2 Luangwa Valley baboons, as well as blood DNA from 2 SNPRC baboons. For each fecal DNA sample, we combined 1–2 μg of extracted fecal DNA with 160 μl of prepared protein-bound beads and a variable volume of ice-cold 5x bind/wash buffer for maintaining 1x concentration of bind/wash buffer. After combining beads and DNA, we incubated the mixture at room temperature with rotation for 15 min. DNA and MBD2-Fc-bound magnetic beads were then collected by magnet and the supernatant removed. For samples in library A, we washed the collected beads with 1 ml of ice-cold 1x bind/wash buffer. For samples in library B, we conducted three expanded wash steps to maximize the removal of unbound DNA. For each wash in library B, we added 1 ml of ice-cold 1x bind/wash buffer and mixed the beads on a rotating mixer for three minutes at room temperature before collecting the beads by magnet and removing the supernatant. Following the final wash, we resuspended and incubated the beads at 65 °C with 150 μL of 1x TE buffer and 15 μL of Proteinase K for 20 min with occasional mixing. The eluted DNA was then separated by magnet, purified with 1.5x homemade SPRI bead mixtureE. coli DNA (K12 or ATCC 11303 strains) at controlled proportions. The resulting post-enrichment proportion of baboon and E. coli DNA was evaluated by qPCR in two analyses using (1) universal mammalian MYCBP36 and (2) universal bacterial 16 S rRNA (16 S)37 primers along with standards created from the same respective organisms , which substantially improved results. Our initial serial enrichment experiments failed to recover DNA, likely due to the use of proteinase K (combined with TE buffer) in the manufacturer’s elution protocol. Hypothesizing that incomplete removal of proteinase K, even following bead cleanup, interfered with the enrichment protocol, we instead eluted DNA using a high (2 M) NaCl concentration, resulting in successful serial enrichment. These changes, along with a full modified protocol, are detailed in the Supplemental Protocol.For our next two libraries, libraries C and D, we added a much smaller volume of prepared MBD2-Fc-bound magnetic beads (1–22 μl) based on the estimated proportion of starting host DNA, kept the capture reaction volume consistent at a relatively low 40 μl (concentrating samples as needed using a SPRI bead cleanup), added an extra wash step in which samples were resuspended in 100 μl of 1x bind/wash buffer then incubated at room temperature for 3 minutes with rotation, and eluted samples in 100 μl 2 M NaCl. For four fecal DNA samples in library C and all of library D, we serially enriched the samples by repeating the capture reaction with 30 μl of MBD-enriched DNA (post SPRI-bead cleanup). Library C included fecal DNA from 5 SNPRC baboons, 2 Kafue National Park baboons, and 1 Luangwa Valley baboon. Library D contained fecal DNA from 6 Lower Zambezi National Park baboons, 4 Choma baboons, and 30 Kafue National Park baboons. We prepared a final library, library E, from independently extracted blood DNA from five SNPRC baboons in order to quantify the stochasticity associated with independent library preparation from independent extracts. The composition of libraries A-E are described in detail in Supplemental Tables 27 with modifications to accommodate low input as described below.Library preparation followed standard double-digest restriction site-associated DNA sequencing (ddRADseq) proceduresSphI and MluCI (New England Biolabs), following a ratio of 1 unit of each enzyme per 20 ng of DNA. Enzymes were diluted up to 10× using compatible diluents (New England Biolabs) to facilitate pipetting of small quantities, using an excess of enzyme if necessary to avoid pipetting less than 1 μl of the diluted enzyme mix. As the total amount of post-enrichment fecal DNA is by nature low, we adjusted adapter concentrations in the ligation reaction to ~0.1 μM for barcoded P1 and ~3 μM for P2, which correspond to excesses of adapters between 1–2 orders of magnitude. Since adapter-ligated samples are multiplexed into pools in equimolar amounts, we made efforts to combine samples with similar concentrations and enrichment when known. We used the BluePippin (Sage Science) with a 1.5% agarose gel cassette for automated size selection of pooled individuals, with a target of 300 bp (including adapters) and extraction of a “tight” collection range (±39 bp). For PCR amplification, we ran all reactions in quadruplicate to minimize PCR biases and attempted to limit the number of PCR cycles. As the concentration of post-size-selection pools was below the limits of detection without loss of a considerable fraction of the sample, estimation of the required number of PCR cycles was difficult. We therefore iteratively quantified products post-PCR and added cycles as necessary. The total number of PCR cycles per pool is reported in Supplemental Table For all samples, including blood DNA and MBD-enriched fecal DNA, we digested DNA with 38. For every pair of blood and fecal samples from the same individual, we downsampled mapped reads to create new pairs with equal coverage in order to control for biases due to differences in sequencing depth. After realignment around indels, we identified variants using GATK UnifiedGenotyper39, in parallel analyses (1) calling variants in all samples at once and (2) processing each sample in isolation to avoid biasing variant calls from other samples at the expense of accuracy. Homozygous sites matching the reference genome were listed as missing when variants were inferred in single individuals. Variants were filtered with GATK VariantFiltration  and indels were excluded.We demultiplexed reads by sample and mapped them to the baboon reference genome  using BWA with default parameters and the BWA-ALN algorithmin silico, tallied reads within each predicted RADtag, and gathered the following information about each region: length, GC percentage, and CpG count in region ± 5 kb. We also calculated read depth in these simulated RADtags. Distributions of blood and fecal RADtags’ length, GC percentage, and local CpG density . Using just the samples that were part of the same-individual blood-feces pairs, we then performed an association test and missingness chi-square test to detect allele frequencies or missingness that correlated with sample type. We did the same with the unfiltered dataset as well. Though we had few pairs of fecal samples from the same individual, we computed distance between pairs of samples from the same individual using the stringently filtered dataset to compare distance between and within sample types via a Wilcoxon signed rank test.To create a stringently filtered dataset with high genotyping rate, we filtered the multi-sample called SNPs in PLINKSupplemental Information


In addition, the multivariate ostracod faunal stratigraphic trend (nMDS axis-1 sample score) is calibrated using bathymetric distributions of extant molluscs sampled from the same stratigraphic intervals along the VdM section. Data and analyses support the research article “Dynamics of benthic marine communities across the Early-Middle Pleistocene boundary in the Mediterranean region : biotic and stratigraphic implications” Rossi et al. [1].Ostracod faunal turnover and oxygen isotope data (foraminifera) along the Valle di Manche (VdM) section are herein compiled. Specifically, the material reported in this work includes quantitative palaeoecological data and patterns of ostracod fauna framed within a high-resolution oxygen isotope stratigraphy (δ Specifications TableValue of the data•Valle di Manche (VdM) is a key-section within the Mediterranean Basin as it straddles the Early-Middle Pleistocene boundary and contains a record of the Matuyama–Brunhes reversal. The abundance data of benthic organisms here presented complement the available documentation for the VdM section.•The multidisciplinary approach adopted provides a viable strategy for quantifying stratigraphic and palaeontological patterns, which allowed for an improved reconstruction of depositional environments.•The data here presented could be compared to other Mediterranean siliciclastic successions that record Early-Middle Pleistocene high frequency sea level fluctuations.1Uvigerina peregrina sampled at high resolution along the 38m-thick investigated interval of the Valle di Manche section  and non-metric multi-dimensional scaling (nMDS) are two widely employed indirect ordination methods in palaeoecology. As both ordination techniques have different strengths and weaknesses, the best approach is to use both methods as a crosscheck on the robustness of the outputs 2.2Reduced Major Axis (RMA) regression was performed to explore the relationship between ostracod and mollusc faunal composition along the Valle di Manche section . The mul2.3U. peregrina were analysed in order to reduce statistical variability. After being lightly crushed, to remove organic contaminants, the selected specimens were soaked in hydrogen peroxide (3%). Then, analytical grade acetone was added, and the samples cleaned ultrasonically, after which the excess liquid was removed. All stable isotope analyses were carried out on an automated continuous flow carbonate preparation GasBench II device, attached to a Thermo Scientific Delta V Advantage Isotope Ratio Mass Spectrometer. Measurements of δ18O were determined relative to the Vienna Peedee belemnite (VPDB) standard, with an analytical precision that is better than 0.1‰.Between 10 and 15 specimens of Uvigerina peregrina δ18O signal to the stacked planktonic oxygen isotope record derived from the Mediterranean Sea 18O signal from the VdM succession to a benthic record from the Mediterranean region appeared to be a more advisable choice than the use of a planktonic δ18O stack as a tuning target. However, the benthic δ18O from VdM and the benthic δ18O stack of 18O stack in the time interval from ca. 860 to 815 ka (MIS 21), lead to difficulties in developing a tuned timescale  is interpreted as a driver of ostracod turnover along sedimentary successions. In this work, sand percentage was plotted against DCA axis-1 sample scores A via linA linear correlation model (RMA) was also applied for bathymetry estimates of ostracod samples B. Given Anomia ephippium, Heteranomia squamula) were excluded from calibration, as they commonly show low association between ordination scores and bathymetry Sample-level bathymetry was calculated via the weighted average of a sub-set of extant mollusc species for which optimum bathymetry values were known  to LC) and University of Bologna .This research was funded by the


Dura, Pisifera and their hybrid progeny Tenera has not yet been well understood. Here we compared the temporal and spatial compositions of lipids and transcriptomes for two oil yielding organs mesocarp and endosperm from Dura, Pisifera and Tenera. Multiple lipid biosynthesis pathways are highly enriched in all non-additive expression pattern in endosperm, while cytokinine biosynthesis and cell cycle pathways are highly enriched both in endosperm and mesocarp. Compared with parental palms, the high oil content in Tenera was associated with much higher transcript levels of EgWRI1, homolog of Arabidopsis thaliana WRINKLED1. Among 338 identified genes in lipid synthesis, 207 (61%) has been identified to contain the WRI1 specific binding AW motif. We further functionally identified EgWRI1-1, one of three EgWRI1 orthologs, by genetic complementation of the Arabidopsis wri1 mutant. Ectopic expression of EgWRI1-1 in plant produced dramatically increased seed mass and oil content, with oil profile changed. Our findings provide an explanation for EgWRI1 as an important gene contributing hybrid vigor in lipid biosynthesis in oil palm.Oil palm is the most productive oil crop in the world and composes 36% of the world production. However, the molecular mechanisms of hybrids vigor (or heterosis) between  Heterosis in plants of diverse species is associated with many superior agronomic characteristics, including larger plant stature, increased biomass, growth rate, grain yield, and tolerance to abiotic stresses. These heterosis is frequently found in cereal crops such as maize (Zea mays) and rice (Oryza sativa), mainly as hybrids, and many other crops including bread wheat (Triticum aestivum), upland cotton (Gossypium hirsutum), and few in oil crop e.g. canola or rape seed (Brassica napus), are grown as allopolyploids. Heterosis has been widely used by breeding program to improve crop yield and resistance significantly. Despite being widely exploited for many decades, the mechanisms underlying heterosis still remain poorly understood esp. for high energy content oil crop5. Most of the earlier efforts focused on genetic analysis of the modes of gene action. Many observations point to the likelihood that the diverse genetic and molecular mechanisms at a large number of genes are responsible for hybrid vigor in any heterotic parental pair. A few classical genetic concepts were previously considered to explain hybrid vigor and the most accepted hypothesis are dominance, the over-dominance and epistatic effects6. Recently, it was suggested that a combination of different genetic principles might best explain the mechanism of hybrid vigor8. However, identifying the specific molecular mechanisms that are responsible for the phenotypic differences between the hybrid and inbred parents remains a challenge, although successes in other plants have been achieved, such as the demonstration of heterosis in yield by the effects of a single gene Single Flowering Truss in tomato9.Hybrid vigor, or heterosis, refers to the superior performance of hybrid progeny relative to their inbred parents6.In recent years, study of heterosis has been approached to the level of the gene products such as transcripts and the regulation of their expression. Available technologies have enabled addressing the biological concerns at the genomic scales, the proteome, the metabolome and especially transcriptome. Various mechanisms may be involved in RNA expression regulation including genetic or epigenetic factors, such as chromosomal structure, DNA sequence diversity, and DNA methylation12, soybean13 and most important oil palm14. Oil palm (Elaeis guineensis Jacq) originates from tropical Africa and it was imported into South Asia where industrial plantations started about 100 years ago. Oil palm is now the most productive oil  crop in the world with an average oil yield of 5–7 tons per hectare15 and contributes about 36% of world oil production. In addition to triglycerides, palm oil also contains high levels of carotenoids and vitamin E, which are believed to counter the ravages of chronic diseases such as heart disease and cancer and to delay aging15.The use of hybrid have been highly promoted in oil crops yield including rape seedTenera palms by crossing of Dura and Pisifera. Crossing Dura and Pisifera to give the hybrid progeny fruit type improved partition of dry matter within the fruit, resulting in a more than 30% increase in oil yield per tree. With the progress of genome sequencing project for parental Dura16 and Pisifera15, it is possible to underpin the molecular mechanisms of hybrid vigor of Tenera. Therefore, this model will be used to understand heterosis in lipid biosynthesis for oil crop. A few efforts of genome-wide gene identification for oil biosynthesis have been done19. Despite its obvious scientific and economic interest, literature and molecular resources available for oil palm remain elusive, esp. for the molecular mechanism of hybrid vigor in oil yield is still scarce. Aside from the SHELL gene that developmentally controls cell type differentiation20, the heterosis in lipid biochemical synthesis and the molecular clues are largely unknown. Furthermore, the chemical and biochemical basis of oil yield heterosis need to be further characterized.Almost all modern, commercial planting material consists of 26.Presently, knowledge of TAG accumulation in plants is mostly based on studies of oil seeds. Sucrose as the main source of carbon for storage oil synthesis in higher plants, is converted to pyruvate via glycolysis in non-green tissues. Pyruvate is the main precursor for the acetyl-CoA molecules destined to fatty acid synthesis. Plastid pyruvate kinase (PK), pyruvate dehydrogenase (PDH), and acetyl-CoA carboxylase are considered as key enzymes for fatty acid synthesis and are regulated by transcription factors in oil seeds, including WRINKLED1 (WRI1)27. We also sequenced Dura genome and did resequencing on elites’ trees16. With the whole genome sequence availability, we here try to globally survey the transcriptome of the two inbred lines Dura and Pisifera in comparison to their F1-progeny Tenera in an unbiased approach via RNA-seq. To identify features specific to high oil production in non-seed tissues such as oil palm mesocarp, we first identified the key lipid biosynthesis stage and generated several billion reads for the mesocarp. At the same time, we did parallel work for seed part of the endosperm. By choosing the particular stages, we intended to identify transcriptional patterns and regulators possibly determining the processes leading to heterosis for the lipid traits. Moreover, we carried out transgenic analysis to identify candidate key genes to be responsible for hybrid vigor in oil biosynthesis of oil palm. The focus of this temporal and comparative analysis was to gain insight into factors responsible for this dramatic difference in carbon partitioning. In addition, a better understanding of oil accumulation in fruits may present strategies for engineering oil accumulation in other vegetative tissues.We have recently identified some candidate gene responsible for tree height in oil palmTenera than parental oil palms Dura and Pisifera. Due to extremely low fertile fruit ratio for Pisifera (only less 10% VS 70% for Dura and Tenera), the low fruit yield palm Pisifera is not commercially used in oil palm plantation except to provide pollen for generating the hybrid progeny Tenera. Further analysis on key lipid traits, for example lipid content per fruit  as indicated in final mature fruit stages (5.5 months after fertilization). Mesocarp of Pisifera commences rapid lipid biosynthesis and accumulation at very early stage, as highly as 10% of total lipid content at 1.5 months after fertilization (MAF), 2 months ahead of Tenera, which is one month ahead of Dura at 4.5 MAF  of upregulated expression gene than downregulated expression gene  and higher  expression levels, were estimated by fold change . A significant higher portion of genes in mesocarp showed nonadditive expression pattern, indicating a remarkable expression changes have taken place in mesocarp than seed organ endosperm Table . We furtA detailed analysis between additive and nonadditive gene expression based on whole genome or individual pathway data generated similar conclusion for both endosperm and mesocarp  Fig. . InteresTenera against these of Dura. As shown in Fig. Dura and Tenera on two tissues , for which the expression was about 50-fold higher in Tenera relative to Dura. Contrary to highly expression of FAS related genes in endosperm, the level of FAS transcripts only mildly increased in Tenera than in Dura in mesocarp, except ketoacyl ACP synthase I (KASI), which encodes the key enzyme for fatty acid elongation, with an increase of 28-fold in Tenera , which encodes the enzyme to convert C16 to C18, with an increase of 11-fold in Tenera. Since the expression changes were found in key genes for fatty acid composition determination, we deduced a higher C18:C16 ratio in Tenera. Gas chromatographic analysis showed that a much higher C18:C16 ratio in mesocarp of Tenera (1.60) than that of in Dura (1.28), consistent with the much higher KASII expression level found in mesocarp of Tenera  than that of in Dura (1.0) (Supplementary Table FAD3), encoding enzyme to synthesis α-linolenic acid C18:3 and longer fatty acid, are highly expressed in Tenera, fitting well with higher C18:3 and further derivate fatty acids in Tenera (3.6% Vs 0.7% in Dura)  that catalyse the last (acyl-CoA dependent) acylation step to TAG, with 8.6-fold and 2.3-fold of Dura in endosperm respectively  enzymes were, on average, 8-fold higher in endosperm of ura Fig. . These eera Fig. . The milera Fig. . The desely Fig. .Tenera , choline-phosphate cytidylyltransferase (CCT) and long-chain acyl-CoA synthesis (LACS)  in Tenera, which may explain the reverse trend of fatty acid composition change (higher poly- VS lower monosaturated fatty acid), compared with that of Dura in both mesocarp and endosperm  membrane based polar phosphatidylchoilne (PC) synthesis pathway was also of highly ectopic expression in era Fig. . SeveralTenera over its parental.In summary, the expression pattern for most of the genes in lipid biosynthesis pathway is consistent with metabolite profiling analysis. Enhanced expression of genes in glycolysis and lipid biosynthesis pathways may be one of the fundamental reasons for the lipid heterosis of Tenera compared with Dura, we suspected that some master regulators such as transcriptional factor may be involved in the transcriptional regulation of lipid heterosis. Three paralogs of the WRINKLED1 (WRI1) transcription factor were identified from our own and other published transcriptome data18. Here, we performed real-time quantitative RT-PCR to analyse the expression profile of three WRI1 genes in various tissues at different developing stages by designing gene specific primers shared by EgWRI1-1 and EgWRI1-2. As shown in Fig. WRI1 coincided well with the oil biosynthesis in mesocarp and endosperm  of mesocarp of Pisifera, which suggested that the early rapid lipid biosynthesis accumulation may due to the early expression of EgWRI1-1 and EgWRI1-3 in mesocarp of Pisifera  has shown to contain WRI1 specific binding AW motif, same with previous findings22. This may also be one of the reasons to explain the important functions of WRI1 in oil yield of oil palm  of up-regulated to down-regulated expression gene was observed in this transcriptome data, implying that the increased efficiency of a subset of transcripts were responsible for the heterosis between Tenera and its parental lines.Although heterosis has been widely exploited in plant breeding and plays an important role in agriculture34. These significant differential genes between Tenera and its parents are derived from the heterozygosis of the combined hybrid genomes and may be potentially associated with phenotypic changes in the hybrid. Although QTL analysis is still very limited because of the narrow number of markers36, We are still able to identify single nucleotide polymorphism (SNP) based on our sequencing data. After mapping SNPs to gene locus, a lot of allele specific genes were identified  polyploids, in which genes derived from the A and D subgenomes show different relative expression levels among different tissue types40. Some people indicated that it may be due to the allele variations that caused these different regulatory expression patterns28.By comparing the allelic expression ratios in mesocarp and endosperm of F1 hybrid plants, we were able to test for expression variation among different tissues. Lipid biosynthesis pathway genes exhibited hybrid vigor in endosperm but not in mesocarp. However, glycolysis did not show the similar pattern. The most likely explanation for the different additively and nonadditively pattern among different tissues is that there are tissue-specific effects on their relative expression levels during crossing and evolution. This finding is similar to studies of the homoeologous genes in cotton , father palm population Pisifera  and the crossing population Tenera oil palm (Elaeis guineensis) trees used in this research were planted in plantation of Wilmar International, South Sumatra . The management and field yield data of the three populations was carried out following the standard protocol of Wilmar International. Dura fruits were harvested from trees from a Dura parent of Deli origin (Dura A), within the same self-progeny of a single tree. Pisifera fruits were harvested from trees from a Tenera X Pisifera parent of TS3 (African AVROS origin) within the same self-progeny of a selfed population. For each stage of development for Dura and Pisifera, at three independent bunches were tagged and harvested on three distinct individuals of the same genotype. For Tenera samples, two bunches of each developmental stage fruits of the tree #53 were tagged. Ten spikelets were then collected from each bunch and all undamaged fruits withdrawn from each of the ten spikelets were randomly sampled. Mesocarp and endosperm of each fruit were separated and flash frozen in liquid nitrogen.Mother palm population http://www.bioinformatics.babraham.ac.uk/projects/fastqc/]. Trinity41 was used for de novo assembly of the raw reads to generate unigenes. Totally 59,078 unigenes was got with around 96,062 isoforms [Supplementary Data Set Arabidopsis thaliana protein annotation database (TAIR 10) was used to the function annotation, with at least one hit in BLASTX search with E-value < = 1e−3. RSEM (RNA-Seq by Expectation Maximization)42 was used for abundance estimation for assembled transcripts to measure the expression level.. Information on data files containing function annotation and temporal expression is provided in Supplementary Data Set Twenty-four RNA libraries were prepared and sequenced by Illumina HiSeq2000, following the manufacturer’s instructions. Around 20 million RNAseq reads of 101 bp length were generated from mesocarp and kernel samples respectively. The quality of the generated sequence was checked by FASTQC [43. The sample discs were rinsed three times in 0.1 M phosphate buffer for 15 min each, and were then post-fixed in 1% (w/v) aqueous OsO4 for 1 hr. Tissues were dehydrated in an ethanol series and embedded in Spurr’s resin. Semi-thin sections with thickness of 500 nm were stained in 0.1% toluidine blue and photographed with a Zeiss Axioplan2 microscope .Dry Arabidopsis seeds were collected and visualized under a scanning electron microscope . For Light microscopy, mesocarp and endosperm discs were excised from fresh oil palm fruit and fixed overnight in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2 as described before44. The complete open reading frame of EgFATB1, EgFATB2, EgDGAT1, EgDGAT2, EgWRI1-1, and EgWRI1-3 was amplified by PCR using the primer pairs list in Supplementary Table Arabidopsis thaliana wild type (Col-0) and homozygous Arabidopsis wri1-4 mutants (Col-0 background) as start plant materials. Sterilized seeds were incubated on Murashige and Skoog medium at 4 °C for 3 d and transferred to long-day conditions for growth (16 h of L/8 h of D). Transgenes were introduced into plants by Agrobacterium tumefaciens–mediated infiltration using the Arabidopsis floral dip method45. For seed mass analysis, seed mass of each plant was determined by weighing mature dry seeds collected from one whole plant.The pBA-002 and pCAMBIA1300-derived vectors were used for all vector construction in this reportTo analyse the lipid content in oil palm, individual tissue and each stage of development, lipid analyses were performed in triplicate (from three different extractions) using a completely random experimental design. Each extraction is from materials of at least 3 fruits samples. The dry mesocarp and endosperm part were grinded to fine powder and the lipids were extracted with hexane three times. The weight of the total lipid was determined and the total lipid content was recorded as the ratio of total lipid to dried endosperm or mesocarp weight . Total lipid content in one year for one tree was calculated as  * average year endosperm or mesocarp yield per tree . The data were presented as means ± standard deviations.et al. described46. FAMEs were generated as described before47 and separated by GC and detected using GC Agilent 7890 coupled with 5975 C MS . The data were presented as means ± standard deviations.For Arabidopsis seeds fatty acid analysis, total lipid was extracted and transmethylated from 100 dry Arabidopsis seeds as Li 48. Quantitative RT-PCR for the genes in this study  or plant RNA purification reagent , following with DNAse treatmentTruSeq RNA Sample Preparation Kits v2 was used to generating mRNA-focused libraries from total RNA according to the protocol instruction.http://www.ddbj.nig.ac.jp/) under accession number DRA001857The RNA-seq data supporting this study are available in the DNA Data Bank of Japan (DDBJ: Supplementary FigureSupplementary Data Set


With this process, entitled “exBAT”, we identified conditions, in both mouse and human tissue, that convert whole fragments of WAT to BAT via a single step and without unwanted off-target pharmacological effects. We show that ex vivo, exBAT exhibited UCP1 immunostaining, lipid droplet formation, and mitochondrial metabolic activity consistent with native BAT. In mice, exBAT exhibited a highly durable phenotype for at least 8 weeks. Overall, these results enable a simple and scalable tissue-grafting strategy, rather than pharmacological approaches, for increasing endogenous BAT and studying its effect on host weight and metabolism.There is widespread evidence that increasing functional mass of brown adipose tissue (BAT) via browning of white adipose tissue (WAT) could potentially counter obesity and diabetes. However, most current approaches focus on administration of pharmacological compounds which expose patients to highly undesirable side effects. Here, we describe a simple and direct tissue-grafting approach to increase BAT mass through  Whereas changes in diet and exercise are often ineffective in practice3, current weight-loss drugs typically exhibit considerable side effects, are contraindicated in many individuals, and either induce only moderate weight loss or are plainly ineffective in many patients5. Bariatric surgeries can induce large weight loss and remission of type II diabetes symptoms in the early months, but the procedures are invasive and can produce complications6, and weight is often re-gained and up to a third of patients who have remission of type II diabetes symptoms relapse after several years7. Thus, there remains an important need to explore new weight-loss approaches with the potential for low rates of complications and long-term efficacy.The obesity epidemic presents significant health and economic risks, afflicting 78 million adults and 13 million children with medical costs estimated to be $150 billion in the U.S. alone28. BAT is a highly metabolic tissue that exhibits thermogenesis in response to cold exposure via uncoupled respiration mediated by the BAT-specific protein uncoupled protein 1 (UCP1)29. BAT consumes large amounts of fatty acids and glucose to support thermogenesis, such that only a small amount of active BAT  can account for 20% of daily energy expenditure30. Humans and animals have two major forms of BAT: native BAT, a permanent form present at distinct anatomic sites  and inducible BAT , which can develop within WAT in response to endogenous or exogenous stimuli, in a process referred to as browning37.Over the past decade, manipulation of brown adipose tissue (BAT) has emerged as a promising approach for treating obesity and metabolic disease14. While several approaches have been proposed for increasing BAT mass or activity in humans, significant drawbacks have prevented them from reaching the clinic. First, cold exposure has resulted in reductions in body fat mass and changes in glucose and insulin sensitivity42, but long-term cold exposure is nearly impossible to implement for most patients. Second, several classes of drugs (such as sympathomimetic drugs and β3-adrenergic agonists) have been shown to increase BAT activity in animals and humans; however, these drugs have been found to exert unwanted off-target effects, including potentially life-threatening cardiovascular effects, limiting their safety and efficacy43. Third, while differentiation of patient-derived adipocyte progenitors for transplantation is a potentially promising strategy to increase BAT mass, this method poses significant manufacturing challenges due to the procedural complexity involved in isolation, expansion, and differentiation of autologous adipose precursor cells for individual patients46. The lack of vascularization and other tissue components may also limit the viability and function of large amounts of transplanted cells47.There is widespread evidence in animals that increasing the mass or activity of BAT or beige fat has enormous potential to prevent and/or reverse obesity and metabolic diseasein vivo, by isolating host WAT, conversion of whole adipose tissue fragments to BAT-like fragments in a single ex vivo step, followed by implantation of the converted BAT. This method is simple to perform, can be easily scaled, eliminates the danger of off-target pharmacologic effects, and retains the native vascularization and tissue microenvironment important in maintaining BAT functionality, and thus enables metabolic characterization of hosts with tissue-grafted converted BAT.In this study, we explore a novel and direct approach for increasing long-term BAT mass 48, large amounts of viable WAT can be obtained by plastic surgeons using well-established harvesting techniques (i.e. liposuction)50. Moreover, compared to traditional processes that involve sorting and purification of isolated progenitor cells followed by long periods of cell-culture expansion, the exBAT procedure is quick, because a single browning step acts on whole tissue fragments to convert WAT to BAT mass, which is ready for direct implantation.The three steps of exBAT are: 1) harvesting of host WAT; 2) exposure of WAT fragments to browning factors via single-step culture; and 3) re-implantation of converted BAT within subcutaneous WAT , isobutylmethylxanthene , T3 (thyroid hormone), indomethacin (COX inhibitor), CL316,243 (β3 adrenoreceptor agonist), and vascular endothelial growth factor (VEGF) (see Methods for details). This single-step cocktail was found to induce browning over a duration of 1–3 weeks ex-vivo while maintaining cell viability, compared to multi-step induction which was previously performed for browning of adipocyte progenitors52.We first developed a mouse model to test whether whole WAT fragments could be converted to BAT ivo Fig. . We firsin vivo, showed that viability after 3 weeks of ex vivo browning was comparable to that of control media under the same duration , consistent with what would be expected of native BAT.To further test the extent to which we could achieve ex vivo culture, we subcutaneously implanted approximately 0.2 mL of autologous converted BAT on the right inguinal WAT depot and extracted the tissue again from the implant site after 8 weeks.Next, we examined whether the BAT-like properties of tissues cultured in browning media would persist weeks after re-implantation. After 3 weeks of ex vivo process retained a WAT-like appearance . Tissues were cultured in the same media and with the same browning factors as previously described for mouse tissues. After 3 weeks of ex vivo culture, we observed that human WAT tissue fragments cultured in browning media developed significant UCP1 expression and smaller lipid droplets .We next sought to investigate whether human subcutaneous WAT could also be converted to BAT with our single-step ets Fig. . Tissuesets Fig. , concordent Fig. , and citent Fig. , a measu58, to minimize variations in stress and weight caused by the multiple surgeries of autologous re-implantation  which made it suitable for needle injection; although epididymal WAT had previously not been as well demonstrated to convert to BAT as inguinal WAT, we found it to brown effectively using our single-step ex vivo method  mouse model to mimic the potential target population in humans. Here, we used allogeneic transplantation, performed in previous DIO mouse modelsion Fig. . To mimihod Fig. . As a cop = 0.056). Differences in VO2 and heat expenditure in mice 1 week following implantation were non-significant  to produce BAT-like tissue. The approach eliminates the need for technically challenging stem-cell isolation and expansion, which can be complex to implement at scale. This single-culture step could be easily implemented into existing autologous fat grafting procedures, and potentially even production within the clinician’s office (as has been achieved for adipose stem cell-based therapies62).One barrier to the use of engineered BAT as a therapeutic for countering obesity and metabolic disorders is the scalability of traditional cell- and tissue-engineering approaches. The exBAT method is simple and practical to perform. The browning factors used in our media comprise approved drugs or endogenous factors , enhancing safety in case of trace amounts following re-implantationin vivo results.While metabolic phenotyping data was only conducted with a single dosage in this study, future variations of procedural modifications with increased dosage variations could be tested. Additional studies could be performed to further assess changes in cell viability over time. Finally, the use of autologous instead of allogeneic implantations, and the use of inguinal adipose tissue may help produce significant 41. Since large amounts of excess WAT can be harvested from overweight and obese patients via traditional fat-grafting procedures (i.e. liposuction), and small amounts of BAT can have a large impact on metabolism, direct ex vivo browning of WAT could generate sufficient BAT mass to test metabolic benefits more thoroughly than this study. Overall, a tissue-grafting approach to increase endogenous levels of brown fat could present a complementary method, alongside current approaches to reduce energy intake (such as appetite suppressing drugs and surgeries) or pharmacology-based approaches to increasing brown fat, in order to address the significant clinical need for effective therapies for obesity and diabetes.For future studies, we note that the amount of native BAT present in humans, correlated with leanness and metabolic health, is in the range of 50 to 100 mL, and an average of 70 mL of native BAT has been shown to significantly increase whole-body energy expenditure, glucose and fatty acid metabolism−1 sodium ascorbate, 1 μM insulin into Dulbecco’s Modified Eagle Medium . The browning media was prepared by adding 1 μM Dexamethasone, 500 μM Isobutylmethylxanthine, 50 μM Indomethacin, 1 μM Rosiglitazone and 1 μM CL316243, and 250 nM triiodothyronine (T3), and 25 ng mL−1 VEGF into control media.The control media was prepared by adding 10% fetal bovine serum (FBS), 1% Penicillin Streptomycin, 20 mM HEPES, 50 μg mL63. Skin was gently lifted and retracted and a portion of the inguinal WAT depot (approximately 0.3–0.5 grams) was lifted with forceps and excised using a scalpel. Tissues were transferred aseptically to tubes containing phosphate buffered saline (PBS) and weighed. The incisions were closed with wound clips and mice were allowed to recover with a pain-relieving drug  administered for two days.All animal procedures were approved by the Columbia University Institutional Animal Care and Use Committee (IACUC) and all experiments were performed in accordance with relevant guidelines/regulations. The mice used in this study were the C57BL/6 strain (Taconic) and were maintained on a high fat diet (Research Diets) starting at 6 weeks of age. Mice were 22 weeks of age at the time of the initial surgery to extract WAT. During surgical procedures, mice were anesthetized with isoflurane and maintained on a warm water circulating pad to maintain temperature. To extract inguinal WAT, a small 1 cm incision was made on the midline of posterior dorsum adjacent to the inguinal depot. The inguinal WAT depots extend from the dorsolumbar region to the gluteal region of the mouse and are anatomically distinct from surrounding tissue and muscle2) were used to incubate the tissues and 12 mL of each type of media was added into each flask. The adipose tissues were incubated in a 37 °C incubator with 5% CO2 and the culture media were changed every two days for a duration of three weeks. After the culture period, a portion of the live tissue fragments were used for viability and mitochondrial staining and another portion of the tissues were fixed in 4% paraformaldehyde (PFA) for 24 hours and stored in PBS for immunostaining. The rest of cultured tissues (approximately 0.2–0.3 grams) were reimplanted into the same mice where they were initially explanted. 8 weeks after reimplantation, the animals were euthanized by carbon dioxide inhalation, and the tissues were taken out and fixed in 4% PFA for 24 hours and stored in PBS. Implanted tissues were able to be identified by the naked eye  using #11 scalpel blades and rinsed 3 times in 30 mL PBS at 37 °C. Tissue culture flasks  to label cytoplasm in live cells, NucBlue Live Cell Stain ReadyProbes reagent  to label nuclei, and MitoTracker Deep Red FM (Life Technologies) to label active mitochondria. Fragments were then rinsed in DPBS, transferred to a glass slide, sandwiched under a coverslip, and then imaged immediately using a Leica DMi6000b widefield fluorescent microscope. For dual live/dead staining, dead nuclei were stained using Ethidium homodimer and all nuclei were stained using Hoescht blue. Cells were imaged using a Leica DMi6000b widefield fluorescent microscope. Manual counting was performed on maximum intensity projection images of z-stacks (10X magnification), using the Leica LAS AF software.Following dissection or after a specified culture period, tissues were rinsed 3X in PBS and subsequently fixed in 10% neutral buffered formalin overnight at room temperature. Tissues were paraffin embedded, sectioned, and stained by HistoWiz Inc . To quantify percent UCP1/tissue area and mean lipid droplet area, we used Aperio Imagescope (Leica Biosystems) to acquire representative 10x images from each IHC stain for WAT and BAT inguinal tissue, and processed those images through automated segmentation and measurement of DAB positive areas using an ImageJ macro (available upon request).et al.64, which was optimized for the best result in our study .Following dissection or after a specified culture period, tissue fragments were fixed in fresh 4% paraformaldehyde solution in phosphate buffered saline (PBS) overnight, then transferred to PBS. The fixed whole mount adipose tissues were stained with anti-UCP1 antibody produced in rabbit , based on the protocol described by Xue Fluorescent images were acquired using Leica TCS SP5 confocal microscopes using LAS AF software (Leica Microsystems). We maintained consistent exposure settings across different samples and acquired wide-area z-stacks across tissue fragments using an automated stage  and all experiments were performed in accordance with relevant guidelines/regulations. The mice used in this study were the C57BL/6 strain (Taconic) and were maintained on a high fat diet (Research Diets) starting at 6 weeks of age. Donor mice (12 weeks old) were sacrificed using COFollowing the injection, mice were transferred to the mouse metabolic phenotyping core facility at Columbia University and allowed to acclimate for 1 week. Mice were kept un a high-fat diet (Research Diets) beginning at 6 weeks of age and maintained on this diet throughout the duration of the experiment. Food and cages were changed weekly and body weights were measured weekly.Comprehensive Lab Animal Monitoring System  was used for metabolic phenotyping at 1, 11, and 15 weeks post-implantation .We used EchoMRI-100H  to measure percent fat mass of the mice . Measurements were taken before placement in the metabolic chambers, after one week in the chambers at room temperature, and after one week in the chambers at 8 °C. This sequence of MRI measurements was repeated each of the three runs in the metabolic chambers .Human subcutaneous adipose tissue samples were obtained under a protocol approved by the Columbia University Medical Center IRB for research use of de-identified discarded tissue from autologous fat transfer procedures  and all experiments were performed in accordance with relevant guidelines/regulations. Patients reviewed and signed informed-consent documents describing the use of their discarded adipose tissue and de-identified data for research into browning of adipose tissue. Samples were obtained from the abdominal region using standard fat harvesting procedures, which involved local injection of tumescent anesthesia for 15 min followed by manual harvesting using a 3 mm cannula and 20 cc syringe. Approximately 6 cc of leftover tissues were retrieved within 4 hours of removal, transferred to a biosafety cabinet, rinsed 3 times with 100 mL DPBS, then transferred to culture. The adipose fragments were approximately 1–3 mm in size and did not require further mincing or processing for culture.33. Protein lysate (8 μg) was used for the citrate synthase activity assay following the manufacturer’s protocol (Sigma CS070). Tissue samples were homogenized in tissue lysis buffer provided with the kits to obtain protein lysates. After centrifugation and removal of the lipid layer, protein concentration was determined using the BCA Protein Assay (Thermo Fisher Scientific Inc.).Citrate synthase activity assays to assess tissue mitochondrial function were performed as described previouslyFor gene expression assays, total RNA was extracted using Trizol (Invitrogen), treated with DNase (ThermoScientific) and reverse transcribed to cDNA (AppliedBiosystems) according to manufacturer’s instructions. Gene expression, normalized to 36B4, was analyzed by quantitative real-time RT-PCR  using the QuantStudio 6 PCR System (ThermoFisher). Primer sequences are available upon request.To perform immunoblot analysis, homogenized tissue was lysed in protein lysis buffer (Sigma) containing protease and phosphatase inhibitors. Standard western blotting was performed using rabbit polyclonal antibodies to Ucp1  and GAPDH . HRP linked Goat anti-Rabbit  was used as secondary antibody. Proteins were detected by chemiluminescence (Roche) and images were acquired using FusionFx (Peqlab).One and two-way ANOVA tests and Bonferroni/Tukey post hoc tests were performed using Graphpad Prism 7 software. Student’s t-test was performed using Graphpad Prism 7 software.Supplementary InformationVideo S1Video S2Video S3Video S4