Post ENCODE, regulatory sRNAs (rsRNAs) like miRNAs have established their status as one of the core regulatory elements of cell systems. However, large number of rsRNAs are compromised due to traditional approaches to identify miRNAs, limiting the otherwise vast world of rsRNAs mainly to hair-pin loop bred typical miRNAs. The present study has analyzed for the first time a huge volume of sequencing data from 4997 individuals and 25 cancer types to report 11 234 potentially regulatory small RNAs which appear to have deep reaching impact. The rsRNA-target interactions have been studied and validated extensively using experimental data from AGO-crosslinking, DGCR8 knockdown, CLASH, proteome and expression data. A subset of such interactions was also validated independently in the present study using multiple cell lines, by qPCR. Several of the potential rsRNAs have emerged as a critical cancer biomarker controlling some important spots of cell system. The entire study has been presented into an interactive info-analysis portal handling more than 260 GB of processed data. The possible degree of cell system regulation by sRNAs appears to be much higher than previously assumed. Initially, the duplex form of sRNAs capable to form complementarity with target RNAs were found regulating the genes post-transcriptionally ,2. BasedHaving this motivation, the present work was done to locate all such potential regulatory small RNAs in human cell systems. The fundamental working ideas have been : (i) recurrence of any given sRNA across large number of experimental samples provides strong evidence for non-random existence of such sRNAs. (ii) For the given samples, if the RNA-seq/transcriptome expression data is available, it is possible to estimate the anti-correlation for expression between a sRNA and its putative targets, multiple times in a re-affirmative manner. Use of proteome abundance data in measuring anti-correlation with the target gene adds further support to this. (iii) Data from interaction sequencing techniques like AGO-HITS CLIP and CLASH provide a very high confidence experimental proof of interactions between the sRNA and targets.For this study, we considered experimental data available for cancerous and normal states for many individuals as well as cancer types. Consideration of cancer for this study has important reasons. It has huge social, health, financial and medical impacts due to being one of the deadliest health conditions. WHO has noted that in year 2012 itself 14 million new cases and 8.2 million cancer related deaths occurred (World Wide Cancer Report 2014) and the rate of cancer is expected to increase by 70% in the coming two decades. Technically, cancer is a state of uncontrolled cell proliferation which becomes malignant. A cancerous cell has capacity to grow on itself, multiply indefinitely where loss of cell cycle control is apparent, resist all signals to stop them as well as oppose cell death and apoptosis, and become capable to migrate anywhere . This wahttp://cancergenome.nih.gov/), GEO, SRA and UCSC CGHUB (https://cghub.ucsc.edu/). RNA-seq and small RNA-seq reads for five conditions were downloaded from GEO and SRA. Complete list of various conditions and sources is available in Supplementary File S1. Expression data for genes and proteins in terms of normalized RPKM/microarray normalized expression and mass spectrometry abundance data were downloaded from TCGA. RPM for sRNAs were calculated using in-house developed scripts. Argonaute CLIP-seq data was downloaded from starBase (http://www.ensembl.org/index.html). To identify the transcriptionally active regions in the genome, FAIRE-seq, DNase-seq and TFBS peaks were downloaded from ENCODE (The ENCODE Project Consortium). Pathways information of each gene was downloaded from Reactome, Wikipathways, BioCyc and KEGG  assembly using BOWTIE with maximum of two mismatches. Mapped reads were also matched to known mature miRNAs downloaded from miRBase version 21 to filter out reads mapping on known miRNAs. To filter out small RNA reads as some random product, two different criteria were applied: (i) only those reads were considered which appeared more than five times in any given experiment, and (ii) at least for two different experimental conditions. All such reads were subjected to target identification. Targets of novel regulatory small RNAs were identified using TAREF  and TargFor the validation of sRNA:targets interactions identified above, targets and partner rsRNAs were searched across Argonaute CLIP-seq data and CLASH sequencing data. Also, Pearson correlation coefficient (PCC) between a target gene's protein level, transcript abundance and small regulatory RNA expression was calculated to further verify functional relevance of these sRNAs. Argonaute CLIP-seq data was available for four Argonaute proteins namely AGO1, AGO2, AGO3 and AGO4. The reported sRNA:Target interactions were scanned through all these Argonautes’ cross linking data. Using the RNA-seq based RPKM values of target genes and RPM values for sRNAs, expression correlation was calculated between the sRNA and its targets. Since protein abundance was normalized and reported in TCGA, the correlation coefficients were calculated using normalized protein abundance and RPM values.http://biodbnet.abcc.ncifcrf.gov/), and Gene Ontology (GO) data was extracted from ENSEMBL. Gene Enrichment Analysis (GEA) of target genes was done using hypergeometric test with Bonferroni correction in R. Enrichr . Similar steps were taken to identify the Dicer (GSE55324) and TRBP (GSM1548746 and GSM1548747) dependent rsRNAs which were scanned in their corresponding CLIP-seq read data. The genomic sequences with 100 bp left and right flanking regions from the start point of the given sRNAs were analyzed for hairpin-loop and miRNA precursors structure support using miREval  and RNAfFigure Total RNA was isolated from cultured cells of all the four cell lines, A549 (Lung Adenocarcinoma), MDA-MB-231 (Breast cancer), HeLa  and MCF7 (breast cancer), using Trizol reagent , according to the manufacturer's instructions. Subsequent purification using an RNeasy Kit  was carried out to remove residual aromatic compounds. One μg of total RNA was used to make cDNA separately for rsRNAs and gene specific qPCR.rsRNA: cDNA synthesis was done using QuantiMir RT Kit  from System Biosciences (SBI) following manufacturer's protocol. Briefly, small RNAs present in the total RNA were tagged by a Poly-A Tail, followed by annealing of anchor oligo-dT adaptor to the poly-A tail. These were then carried forward for cDNA synthesis resulting in pool of anchor-tailed small RNAs. cDNAs were checked by end-point PCR with kit supplied universal reverse primer and miRNA-specific forward primer (human U6 used as control). The cDNAs were diluted 1:20 before being used for qPCR.Gene: Total RNA was converted to cDNA using random primers and High Capacity cDNA Reverse Transcription  as per manufacturer's instructions. The resultant cDNA was diluted 1:10 for qPCR, subsequent to check with a control β-actin primer.® FAST qPCR Kit (KAPA Biosystems) on a Roche LightCycler® 480 instrument (384-well), using default parameters. HPLC purified primers were synthesized from Sigma-Aldrich for this work. Primers used for qPCR of rsRNA and gene are listed in Supplementary File S2.qPCR was performed using KAPA SYBRSmall RNA reads from all considered cancerous and normal tissue-specific conditions were pooled to identify the unique reads. These unique reads were mapped to human genome assemblies . A total of 523 540 761 unique reads were obtained from sRNA-seq read data, of which 317 890 669 (60%) reads mapped across the human genome with 100% match. The number of reads were reduced to 23 369 669 (7%) when only those reads were considered which were expressed in two or more conditions. 10 789 006 (45%) unique reads out of 23 369 669 unique reads were such reads which exhibited abundance of at least five reads. These 10 789 006 small RNA reads were considered as the final set of small RNAs for further analysis. These sRNA were used to identify targets using TAREF . A totalAs mentioned above, 17 612 different targets (unique genes) were predicted commonly by TargetScan and TAREF systems. In order to validate these target:small RNA interactions, the interactions were first searched against the Argonaute sequencing data downloaded from starBase (version 2) . A totalTo add another layer of validation for their existence and their possible regulatory presence, correlation between the expression patterns for the identified interacting rsRNAs and their target partners was assessed using RNA-seq as well as protein abundance data. Protein abundance data was available for only 192 genes in TCGA for different cancerous conditions whereas gene expression data from microarrays and RNA-seq was available for 17 488 and 17 814 unique genes, respectively. Since sRNAs suppress the expression of the target gene, an anti-correlation was expected between the targeting rsRNA and target. For some cancer conditions either the expression data was not available or the data was restricted by TCGA. Protein and miRNA expression anti-correlation estimation was performed for 4776 patients for 15 cancer conditions for which data was available. Targets validated using protein:sRNA co-expression identified many important small regulatory RNAs which target genes involved in important functions such as tumor enhancer or tumor suppressor genes (Supplementary File S4). Out of 192 genes having protein abundance data, significant anti-correlation for expression was observed with 131 unique genes as the targets for 9446 rsRNAs.P-value < 0.05) and high inverse correlation (PCC > |–0.5-> –1|) coefficient between target genes and targeting rsRNAs was observed for 11 234 sRNAs and 17 093 unique genes for sequencing data, while the microarray data showed 8931 sRNAs having strong anti-correlation with 16 020 target genes. In order to assess if targeting could be associated with anti-sense phenomenon, from the list of targeting rsRNAs those rsRNAs were identified whose biogenesis locus was coinciding with the binding site in the target gene and were originating from the opposite strand of the target. From this analysis, a total of 1185 unique rsRNAs were identified regulating 211 unique genes . Also, it was found that 10 968 rsRNAs targeted UTRs of 13 264 genes with a total of 932 266 interactions.Another level of validation was obtained by performing sRNA and target genes expression analysis using their read counts. A total of 3013 cancerous and normal tissue based experimental conditions were considered. For some cancer tissues the RPKM based gene expression data was not available. In such situation the microarray expression data from TCGA  was used. Details of s/RNA-seq and microarray based expression study are given in Supplementary File S5. Significant  displayed strong inverse correlation. For the remaining pairs for which strong anti-correlation could not be found, it might be plausible that they show such regulation in different tissue origin cell lines, tissue per se or there could be multiple targeting sRNAs involved. For some cases, the target genes were expressing in less than 3 cell lines, making it insufficient for correlation measure. Importantly, we found that most of the rsRNA : target gene pairs exhibited inversely correlated Ct values in the same background of the tissue in which they were discovered. Few prominent examples being rsRNAs 7906, 9345, 4150, 3790, 3091, 2734 and 1336. All these rsRNA were discovered in the lung tissue and they showed lower expression in A549 whereas the target genes were expressed in higher quantity. Same goes for rsRNA 8294 and its target gene expression in breast cancer cell line MCF7. We also observed that rsRNA 3091 which showed expression in breast cancer in addition to lung, was found to reflect the same pattern of anti-correlated expression in MCF7, but not in MDA-MB-231. This highlights the possible role of additional factors leading to rsRNA-target regulation. The importance of tissue background of expression is also highlighted by non-directional levels of rsRNAs 9338 and 551 with their target genes. Overall, the experimental results elucidate the existence and differential expression patterns of the queried rsRNAs and their corresponding targets, suggesting their possible regulatory roles in tissue-specific manner , MDA-MB-231 (Breast cancer), HeLa  and MCF7 (Breast cancer). qPCR confirmed differential expression levels for 19 out of 22 rsRNA and 14 out of corresponding 22 target genes across the cell lines. Rest did not work experimentally under the condition tested. One exception is t-tests between cancer v/s normal conditions gave consistently significant P-values (P < 0.05) for this observation, suggesting the sRNA profile originating from Alu differ significantly between cancer and normal states. Previous studies as well as structural analysis of Alu suggest that they are potent to generate siRNAs through Dicer like endonuclease activities. The association of Alu derived sRNAs with AGO proteins and CLASH data found in this study supports the regulatory roles of these sRNAs and reasons for the observed non-random pattern of Alu derived sRNAs. In the absence of high number of individual samples, such sRNAs would be otherwise rejected as a random product or sequencing artifact. However, with consistent evidence of similar and distinctive patterns across many individual sequencing data for normal and cancer states it is tough to dismiss such sRNAs as random products. With the availability of high throughput sequencing data, some recent studies have already reported the display of similar kind of non-random pattern of sRNA biogenesis from other non-coding RNAs exhibiting regulatory roles in cancer conditions  with their corresponding targets. The functional enrichment analysis over their targets provided us the insight that most of the targets of these regulatory small RNAs were associated with cell development, its life cycle, growth and apoptosis (Supplementary File S8).In the present study, several potential regulatory small RNAs were found significantly differentially expressed between normal and cancer states. The differential expression was evaluated across large number of individual samples, followed by http://14.139.59.221/∼scbb/support_site/all_data/rsRNA-9881-n.html) describes the regulatory implications of regulatory small RNA 9881 and expression relationships between rsRNA-9881 and its targets. An interaction network was built with these 20 target genes to understand their relative standing in the networks. Certain genes emerged as central to several critical processes. The most connected genes were CASP8, OLA1 and SUV39H2. In overall, all these 20 genes were found closely associated. CASP8 is critical for MAPK signaling cascade, apoptosis modulation, insulin signaling and tight junction formation. DDX52 associated genes were found enriched for pathways in cancer, MAPK Signaling and apoptosis. FKBP associated genes were found enriched for genes involved in multiple cancer pathways, insulin signaling, B-cell receptor signaling, focal adhesion, WNT signaling pathways. Adipocytokine signaling, insulin signaling, cancer related pathways, EBVL MP1 signaling, TOR signaling were found enriched in MAP3K connected genes. The MBD4 associated genes had broad range of impacts, affecting FAS pathways, apoptosis, cell cycle, notch signaling, pathways in cancer, glycine-serine-thronine metabolism, cysteine-methionine and other amino acids metabolism. MBD4 has already been reported as a gene which is omnipresent as a tumor suppressor gene whose down regulation is related to several types of cancer. The genes associated with MECR were found enriched for adipogenesis, adipocytokine signaling, fatty acid biosynthesis, PPAR signaling and pathways in cancer. MECR associated genes have been found affected in cancer while MECR is reported as the last step gene of fatty acid biosynthesis pathway, seated at mitochondria, but also standing as the final connecter to the nuclear signal through PPAR  where a user could delve into the details. The portal has been built using next generation web development package JS, D3, Python, PERL, JSON, HighCharts and MySQL, installed over a Linux server. A user can browse the portal through several entry points: by searching for terms, browsing for rsRNAs or by directly opting for the various result sections which in turn transfer the user to entire tool sets, look into the pathways details, and select various cancer states. A wall page contains clustering of the rsRNAs on the basis of seed similarities, which provides dynamic image system and enrichment analysis for the targets of the selected clusters and sRNAs. The provisions of dynamic expression analysis is given where any number of experimental conditions could be selected along with sRNA of choice and its targets. Search can be performed using single term as well as in batch mode. Search results may be explored with two options: Annotation Viewer and Expression Search. On selecting Annotation Viewer, an annotation chart with visual genomic representation of the selected query is displayed. From here, all information for a target site and rsRNA can be accessed. Whereas, selecting expression chart button redirects the user to multiple selection input option page. From the page user can select multiple filters like support type, cancer type, sample type, number of individual and user choice plot selection . The rsRNA browser section facilitates several searches and analysis at single place, where a user can look for a particular rsRNA, its experimental evidences, perform on the spot target and associated expression analysis, their enrichment for GO and pathways, and perform up to three step network visualization. At the browser provisions have been made to download the sequences. Highcharts and Highstock library were implemented for expression analysis chart, Scribl library for Annotation viewer and Linkurious based on SigmaJS library was used for network creation and visualization. Also, a user can perform sequence based search using two different search approaches: (i) Suffix tree based Bowtie search with mismatch provisions, and (ii) BLAST search.One of the important points of this study has been the amount of high throughput data considered to identify regulatory small RNAs and their impacts. While doing so, various experimental data from different platforms were pooled together, analyzed and were related to each other in a meaningful manner. Handling such data and pull out meaningful interpretations require a proper structuring and representation of the data and information tools. In this regard, a number of visually rich and useful representations have been made available as a complementary material at the associated portal  GO bubble chart or table, (B) user gets redirected to respective page where the genes or rsRNAs involved could be selected. On the selection of a gene or rsRNA of interest a final plot opens (C) which shows expression of the target genes and targeting rsRNAs. From the list of genes and rsRNAs only the elements selected by the user are displayed. Besides these all, the portal also contains the details of various cluster analysis, from where again every individual sRNA could be picked and analyzed in depth.http://scbb.ihbt.res.in/SCBB_dept/Software.php.In overall, this information portal provides a huge volume of experimental data in high quality visualization and representation to facilitate the user to have meaningful insights. All supporting materials of this study have also been hosted there. This portal will be also available at


Anderson type II odontoid fractures are reported to be the most common injury of the odontoid process in patients over the age of 65. However, atraumatic occult Anderson type III odontoid fractures have been rarely described and remain a diagnostic challenge. In the present report, we illustrate a 78-year-old female with osteoporosis-associated marked thoracic kyphotic deformity who developed atraumatic Anderson type III occult odontoid fracture and raise awareness of this condition. Anteroposterior and lateral standard radiographs of cervical spine failed to disclose odontoid fracture. Magnetic resonance imaging demonstrated intensity changes of the axis. Subsequent computed tomography clearly demonstrated Anderson type III odontoid fracture. Conservative treatment achieved complete bone union without neurological deteriorations. At 3-year follow-up, the patient was doing well without neurological and radiological deteriorations. Even if the patients have no traumatic event, we have to keep odontoid fractures in our mind as one of the differential diagnoses when we encounter elderly patients with neck pain, especially in patients with osteoporosis-associated marked thoracic kyphotic deformity. Odontoid fractures are caused by minor trauma like falls from standing or seated height in elderly individuals . Type IIIn the present report, we illustrate a unique case of elderly female patients with occult Anderson type III odontoid fracture without any traumatic event. We emphasize the importance of an awareness of this condition and discuss the likely mechanism of the injury in the present case.T-score: −3.5) was significantly low.A 78-year-old female experienced sudden severe neck pain when she looked up the refrigerator  to open its door and visited our institution. She had a mild occasional neck pain before this event; however she reported no traumatic event just before the onset of this symptom or in the past. Physical examination revealed the absence of neurological compromise. In turn, anteroposterior and lateral standard radiographs of cervical spine revealed cervical degenerative spondylosis, but we could not detect apparent fracture Figures . LateralShe complained about persistent severe diffuse neck pain. Four days later, magnetic resonance (MR) imaging was performed to rule out latent fresh compression fracture and overt intensity changes of axis, which was low intensity on both T1- and T2-weighted images but high intensity on STIR image, were found Figures . SubsequAfter the careful explanation of the risk of nonunion/malunion and neurological deteriorations, the patient refused surgical treatment as well as Halo-vest fixation. Conservative treatment with Philadelphia type cervical caller was indicated. Two-month conservative treatment with cervical caller achieved good pain relief. Follow-up CT after 6 months from the initial treatment revealed significant bone union Figures . No appaAnderson and D'Alonzo classified odontoid fractures into 3 types based on the localization of the fracture line passed through . Type I Several studies revealed that, compared with patients younger than 65 years, elderly patients require lower energy, that is, fall from standing or seated height, to fracture their cervical spines –7. AtrauOdontoid fractures are mostly induced by nonphysiological flexion, extension, or rotation force of the upper cervical spine; however exact mechanism is not fully determined , 11. SenThe precise mechanism of odontoid fracture in the present case remains unknown; however significant osteoporosis might play a pivotal role on our case. In addition to significant osteoporosis, we advocate the possible influence of sagittal malalignment of the spine on odontoid fracture in this case. Namely, a compensatory hyperlordosis of cervical spine due to marked thoracic kyphotic deformity might yield routine hyperextension stress to the cervical spine. In turn, senescent degenerative changes make the mid- to lower cervical spine stiffer and shift the motion segment to the upper cervical spine . These aStandard radiographs are routinely used as a screening tool for evaluating cervical spine injuries; however they could not always detect them , 12, 13.The optimal management of odontoid fractures in the elderly population remains unsettled and management of this group of patients is complicated by multiple comorbidities , 14–16. In conclusion, even if the patients have no traumatic event, we have to keep odontoid fractures in our mind as one of the differential diagnoses when we encounter elderly patients with neck pain, especially in patients with osteoporosis-associated marked thoracic kyphotic deformity.


A high prevalence of obstructive sleep apnea (OSA) is seen in edentulous individuals. Treatment options for edentulous OSA patients however are limited with continuous positive airway pressure therapy (CPAP) remaining the current therapy of choice. As CPAP is associated with high non-adherence rates and oral appliance therapy requiring sufficient dentition, there is a clinical need for effective treatment strategies aimed at edentulous OSA patients. The purpose of this study was to present a thorough overview of the literature regarding (1) the effects of nocturnal denture wearing on OSA, (2) the outcomes of oral appliance therapy, and (3) surgical treatment in edentulous OSA patients.A computer-assisted literature search was performed in the MEDLINE database on “edentulism” and “obstructive sleep apnea.” The search yielded a total of 34 original articles.A total of 20 studies were included after exclusion of non-relevant, duplicate, and non-English publications, comprising 4 randomized clinical trials, 12 case reports, and 4 cohort or cross-sectional studies. The outcomes of these studies were addressed in detail concerning nocturnal wearing of dentures, oral appliance therapy, and surgical treatment.Currently, there is no consensus in the literature on the effects of nocturnal wearing of dentures on OSA. Several studies report the successful use of oral appliance therapy, including implant-retained mandibular advancement devices (MADs), in selected cases of edentulous patients with varying stages of OSA. Little evidence is available regarding outcomes of surgical procedures in edentulous patients. Based on the results of this literature overview, the paucity of effective evidence-based treatment strategies for edentulous OSA patients indicates the further need of clinical studies to improve clinical management. Obstructive sleep apnea (OSA) is a major medical problem, estimated to affect up to 15–30 % of male adults and up to 5–15 % of female adults . It is cThe underlying pathophysiology of OSA may vary by age, with increased upper airway collapsibility being a common cause in older patients . PreviouCurrent guidelines propose non-surgical treatment options including lifestyle adjustments, position therapy, and  continuous positive airway pressure (CPAP) as first-line therapy. Second in line are treatments with oral appliances or surgical approaches based on the degree of severity and etiology . EdentulAs treatment of edentulous OSA patients can be challenging, all available treatment options should be considered to ensure optimal management and prevent possible life-threatening complications. The present study aims to provide a thorough overview of the literature regarding (1) the effects of nocturnal denture wearing on OSA, (2) the outcomes of oral appliance therapy, and (3) surgical treatment in edentulous OSA patients.A computer-assisted search was performed in the medical database MEDLINE (from January 1966 to March 2015). A combination of the following medical subject headings (MESH) were used in the search: “Sleep Apnea, Obstructive,” “Jaw, Edentulous,” “Jaw, Edentulous Partially,” and “Mouth, Edentulous.” Inclusion criteria consisted of studies reporting clinical outcomes on treatment of OSA in either partially or fully edentulous patients. Clinical outcome parameters were considered relevant when comprising results from polysomnography studies (PSG) or standardized sleep-related questionnaires. Excluded were studies not reporting on partially or fully edentulous patients, studies lacking relevant clinical outcomes, review articles, non-original studies, and articles written in other languages than English. Potential relevance of the articles was determined by studying their titles and abstracts. When considered relevant, full texts were obtained and read; other studies were included by screening the reference lists of articles that were found during our original search. Relevant data regarding patient characteristics, treatment, and clinical outcome including age, gender, body mass index (BMI), polysomnography results, and questionnaire results were extracted. Included studies were appraised according to the Oxford Centre for Evidence-Based Medicine levels of evidence  levels, as well as a reduction of the retropharyngeal space. Wearing complete dentures overnight showed to improve both AHI and SaO2 levels and resulted in an increase in the retropharyngeal space.With a study of six edentulous OSA patients, Bucca et al.  were the2 was seen in both OSA and non-OSA patients when wearing complete dentures during sleep. These positive effects appeared to be more present in non-OSA patients, suggesting the lack of dentition might play an essential causal role in the AHI increase in these individuals. CE performed in supine position displayed an increase in retropharyngeal space when patients were wearing their dentures. Also, a significant decrease in measured exhaled nitric oxygen (eNO), a marker for inflammation, was found in patients after sleeping with dentures compared to sleeping without dentures. Though the exact role of inflammation in the presence of OSA is still unclear, previous research revealed that pharyngeal inflammation was present in patients with OSA [Similar findings were found in a larger clinical trial evaluating PSG results and cephalometric exams (CE) of 48 edentulous individuals with complete dentures . In thiswith OSA , 25.2 levels in patients wearing dentures overnight compared to no denture wearing.The studies mentioned above performed by Bucca et al. , 12 indiArisaka et al.  also repSeveral non-experimental studies have examined the relationship between nocturnal wearing of dentures and OSA as well. In a cross-sectional study of 58 elderly using questionnaires and portable sleep recordings, Endeshaw et al.  found a Oral appliance therapy is considered an effective treatment option for patients with mild to moderate OSA and a feasible alternative for patients intolerant to CPAP treatment , 32. AmoDue to a lack of dentition to support and retain a MAD, these devices are considered unsuitable for edentulous OSA patients. If sufficient dentition is available, other contraindications to consider include temporomandibular joint (TMJ) dysfunction, severe dental mobility, and periodontal diseases , 21. WheTongue retaining/reposition devices (TRDs) do not require support from dentition and can be used as an alternative in patients lacking sufficient dentition. TRDs use suction forces to hold the tongue in a forward position without mandibular repositioning and have been reported to have a similar performance to that of MADs . Deane eSeveral case studies report the use of oral appliances in both partial and completely edentulous patients with OSA. Eskafi et al.  performe2 levels. A 2-year follow-up showed no deterioration in periodontal and gingival health. Ogawa et al. [2 levels. No problems were reported regarding retention or stability of the device.Giannasi et al.  applied a et al.  also repa et al.  used MAD2 levels [2. No difficulties regarding stability and usage of the device were reported.The use of MAD therapy in a completely edentulous patient with severe OSA refusing CPAP treatment was reported by Piskin et al., resulting in a large improvement of AHI (98 vs. 15/h) and SaO2 levels . A modif2 levels  also rep2 levels  used a s2 levels was notable in all 5 patients who completed follow-up, and treatment resulted in eliminating the OSA (AHI < 5) in 4 patients. Wearing discomfort was reported in only 2 patients due to excessive pressure of the MAD on the labial mucosa in the maxilla. The authors suggested to resolve such issues of discomfort with additional implant placement in the maxilla. In the single patient that underwent such a secondary treatment, complaints of wearing discomfort were resolved. This pilot study indicates that implant-retained MAD therapy could be a viable treatment option for completely edentulous OSA patients by evading poor retention and wearing discomfort.The use of dental implants has been proposed to improve retention, as poor device retention remains a major obstacle of oral appliance treatment in edentulous patients , 32. Hoe2 levels. No problems occurred during the 8-month follow-up; however, the authors did mention the risk of loss of the ortho-implants due dynamic forces.A different intraoral approach was suggested by de Carlos et al. , involviSeveral soft tissue surgical procedures like uvulopalatopharyngoplasty (UPPP) and radiofrequency ablation (RFA) are available for OSA patients, although accompanied by varying success rates , 52. In 2 values. A more recent case study of Sencimen et al. [2 levels.More than two decades ago, Alvarez et al.  reportedn et al.  also repPrevious studies indicate that edentulous individuals are at high risk of developing OSA , 14. WhiAlthough the variance in the use of portable sleep recordings instead of in-laboratory PSGs among studies might have played a role in the conflicting study results, several studies confirm that ambulant PSGs are a reliable alternative for in-laboratory PSGs , 57.Despite the fact that the majority of edentate patients wear their dentures overnight , it is gVarious treatment options have been proposed to treat OSA, ranging from oral appliance therapy to surgical interventions . MorpholIt should be noted that OSA patients in general have an increased risk of postoperative complications including postoperative desaturation, respiratory failure, postoperative cardiac events, and ICU transfers . These gUnfortunately, there is a lack of clinical trials assessing the application of OSA therapies in edentulous patients. In order to improve clinical management, future studies in the form of randomized controlled trials (RCT) that assess the potential of implant-retained MAD therapy and other inventions are crucial. Adequate treatment is of key importance in an effort to reduce potentially serious complications and improve both the overall health and quality of life of these usually elderly patients , 65, 66.Currently, there is no consensus on the effects of nocturnal denture wearing on OSA. The use of modified MADs in edentate patients has been reported successful in selected cases. By avoiding poor retention, implant-retained MADs seem to be a viable treatment alternative to CPAP in the treatment of edentulous OSA patients. Few studies demonstrated the outcomes from surgical treatment in edentulous OSA patients. The overview of the literature presented in this paper demonstrates the paucity of effective evidence-based therapeutic strategies for edentulous OSA patients and the need for further clinical studies to improve clinical management.


Genetic heterogeneity was evaluated with observed heterozygosity (HO). Best-fitting spatial autoregressive models were identified, using socio-demographic variables as covariates. Spatial analysis included surface interpolation and geostatistics of observed and predicted patterns. Prediction accuracy was quantified. Spatial autocorrelation was detected for both socio-demographic and genetic variables. Augsburg City and eastern suburban areas showed higher HO values. The selected model gave best predictions in suburban areas. Fine-scale patterns of genetic heterogeneity were observed. In accordance to literature, more urbanized areas showed higher levels of admixture. This approach showed efficacy for detecting and analyzing subtle patterns of genetic heterogeneity within small areas. It is scalable in number of loci, even up to whole-genome analysis. It may be suggested that this approach may be applicable to investigate the underlying genetic history that is, at least partially, embedded in geographic data.Aiming to investigate fine-scale patterns of genetic heterogeneity in modern humans from a geographic perspective, a genetic geostatistical approach framed within a geographic information system is presented. A sample collected for prospective studies in a small area of southern Germany was analyzed. None indication of genetic heterogeneity was detected in previous analysis. Socio-demographic and genotypic data of German citizens were analyzed (212 SNPs;  Accurate assessment of genetic heterogeneity is relevant to manifold fields, ranging from clinical research, pharmacogenetics, and statistical genetics, over forensic sciences up to evolution . In plaUncovering the genetic basis of complex traits remains an immense and urgent challenge in genetic epidemiological research. Great efforts are set to establish well-designed cohorts and large control samples, intended to serve as basis for genetic epidemiological studies. Besides restricting recruitment to individuals of uniform ancestry, a common strategy applied to efficiently gain a representative sample of the inspected population and to control for potential unknown population substructure is to collect samples in smaller geographical areas, usually in medium to large urban centers e.g., –9)..9]).Even well-characterized or supposedly homogeneous regions may still account for subtle genetic structure with potential geographical components , 10. SloMost available well-standardized methods in geographical genetics  were devWith the aim of unveiling modest amounts of population substructure in a small, admixed area we (a) searched for subtle patters of genetic heterogeneity and (b) explored potential predictors of the observed patterns. To this end, we combined statistical genetics with spatial statistics (geostatistics) within the framework of a Geographic Information System (GIS). A GIS provides a computational environment designed for spatial analysis of geographic data, therefore the most suitable framework to detect, to model, and to analyze the geographic variation of genetic diversity.E − 5% heterozygotes deficit) measured in terms of FIS values , HO ∈ .In order to run HO values were created for the following data sets: HO(ALL), and HO(GER). Interestingly, since the KORA S4 genotypes conform a control population pool for genetic studies ).ion (cf. ).Taking these findings of the KORA S4 sample altogether we can state that fine-scale spatial genetic variation may be assumed in the study area. Our results indicate that patterns of genetic heterogeneity can be present in small regions within Germany. In conclusion, it may be stated that the presented genetic geostatistical approach has the potential of being a powerful tool for detecting, modeling, and analyzing spatial patterns of genetic heterogeneity even within populations inhabiting small regions.Supplement 1 describes the KORA S4 sample.Supplement 2 presents additional analysis of spatial genetic differentiation based on conventional measures.Supplement 3 presents an analysis of genetic differentiation between urban and peri-urban areas.Supplement 4 provides a sensitive analysis of the effect on genetic variation of immigrants in the KORAS4 Survey.Supplement 5 summarizes results of additional multivariate analyses of spatial genetic differentiation.Supplement 6 describes the KORA S4 marker set.Supplement 7 provides an analysis of genetic relatedness of individuals included in the KORAS4 sample.


Enterobacteriaceae.Shigellosis trends in these communities provide insight into global movement ofdrug-resistant  Enterobacteriaceae. OrthodoxJewish communities (OJCs) are a known risk group for shigellosis; Shigellasonnei is cyclically epidemic in OJCs in Israel, and sporadic outbreaksoccur in OJCs elsewhere. We generated whole-genome sequences for 437 isolates ofS. sonnei from OJCs and non-OJCs collected over 22 years inEurope , the United States, Canada, andIsrael and analyzed these within a known global genomic context. Through phylogeneticand genomic analysis, we showed that strains from outbreaks in OJCs outside of Israelare distinct from strains in the general population and relate to a singlemultidrug-resistant sublineage of S. sonnei that prevails in Israel.Further Bayesian phylogenetic analysis showed that this strain emerged approximately30 years ago, demonstrating the speed at which antimicrobial drug–resistantpathogens can spread widely through geographically dispersed, but internationallyconnected, communities.Shigellae are sensitive indicator species for studying trends in the internationaltransmission of antimicrobial-resistant  Enterobacteriaceae are recognized as aglobal public health threat  Shigella spp., the distribution of S.sonnei makes it particularly relevant for studying international transmissionbecause it is the most commonly isolated species in middle- to high-income nations    and explored the possible epidemiologicrelationship of outbreaks in OJCs outside of Israel and endemic shigellosis in Israel. Wealso sought to determine the relationship of these epidemiologic processes with AMRdeterminants in S. sonnei.We generated whole-genome sequences from >400 clinical isolates of S. sonnei isolates as partof this study. These isolates were from patients associated with OJCs outside of Israel(n = 171), from 221 patients in Israel , or frompatients in the United Kingdom not associated with OJCs (n = 45)  . The iso(n = 45) . The colh agency .S. sonnei in Israel werecollected during 1992–2014 in local hospital and health maintenanceorganization laboratories, including the national sentinel laboratory-basedsurveillance program  derived from OJCs . These samples included 22 from a small OJC-associated outbreak    (16). Inhttp://www.ebi.ac.uk/ena; accession numbers in DNA was extracted at multiple sites by using the Wizard genomic DNA extraction kit according to manufacturer’s instructions. DNA wassequenced by using the MiSeq and HiSeq 2000 platforms  atmultiple institutes according to in-house protocols  (25–27).S.sonnei Ss046 (GenBank accession no. CP000038), then masking mobile andrepetitive elements  was used to estimate the emergence date ofthe lineage  located outside theOJC-associated lineage, distributed elsewhere in the global III and II lineages . UltimatConsistent with this finding, phylogenetic relationships within the OJC-associatedlineage were defined more by time than geography . Since tS. sonnei, we determined the AMRcharacteristics of the OJC-associated lineage. We found that the lineage had a uniqueAMR profile relative to isolates outside of the OJC-associated lineage . This region is identical to a Tn7-like island, also adjacent to theglmS gene, in the newly emerging Xv serotype of S.flexneri   Figure 2eblaTEM  Figure 2ul1 gene  Figure 2ul1 gene  Figure 2plasmids Figure 2 plasmid Figure 2lineage ; these glineage  Figure 2S. sonnei and its AMR determinants amongOJCs over several decades. These analyses offer insight for the epidemiology ofshigellosis inside and outside of Israel as well as for the broader transmission of AMRenteric pathogens. We showed that, in countries outside of Israel, outbreak strains inOJCs were distinct from strains circulating in the general population and thatOJC-associated strains were more closely affiliated with outbreaks associated with OJCsin other countries (irrespective of geographic distance) and strains circulating inIsrael. Strains from Israel and strains from nearly all previously reported OJCoutbreaks elsewhere formed a distinct OJC-associated sublineage that emerged ≈30years ago. Unlike other described emergent Shigella sublineages    were alsS. flexneri Xv (S. flexneri 3a among men who have sex withmen or of S. sonnei in Vietnam, further acquisition of AMR does notappear to have shaped the subsequent evolution of the lineage. The presence ofadditional resistance genes (S. sonnei that supports itsmaintenance in these communities. This likelihood is consistent with previous studies ofOJC-associated shigellosis, which suggest that transmission is largely driven bycommunal childcare arrangements in a host population that is young and denselystructured (The acquisition of the Tn7/Int2-encoded MDR in this lineage may have facilitated itsepidemic emergence, as has been hypothesized for the broader global III lineage (ce genes  was notThis study documents the speed with which MDR enteric bacteria can transmitintercontinentally through travel within a geographically dispersed, but closely linked,community. Awareness of this mode of sustained, geographically noncontiguoustransmission must inform public health practice, including targeted control andreduction of consequences of the pathogen through developing effective relationshipswith affected communities; identifying specific risk factors; and designing, piloting,and eventually implementing specific culturally appropriate interventions with theparticipation and support of the community while avoiding stigmatization. Effectiveongoing surveillance is also vital, and we demonstrated the resolution required for thatpurpose and the need for effective data sharing to track these otherwise silenttransmission phenomena. Furthermore, the repeated application of high-resolution toolssupports identification of parallels and contrasts with previous genomic epidemiologystudies, supporting construction of a complete picture of the global transmission of AMRenteric pathogens, advancing our position for tackling this global public healthissue.Shigellasonnei used in this study and reference strains from globalcontext database.Sample details for isolates of Shigellasonnei.Supporting data and phylogenetic and genetic characteristics of isolates of


Streptomyces canus ATCC 12647, a producer of the antibiotic telomycin, noted for its unique antibacterial activity against cardiolipin. Genomic analysis using the bioinformatics tool PRISM revealed the presence of multiple biosynthetic gene clusters, including those for telomycin and other natural products of potential pharmacological interest.We present here the genome sequence of  Streptomyces spp. are well known for their plentiful biosynthetic gene clusters, which encode a wide array of small molecules produced through modular assembly line enzymes. Streptomyces canus C-509 (ATCC 12647) is a notable member of this group and was previously isolated by researchers of the Bristol-Myers Company (now Bristol-Myers Squibb Co.) in the 1950s, who identified its production of telomycin, an antibiotic with noteworthy bactericidal activity  using a previously described protocol (Genomic DNA was obtained from the cultivation of protocol . Bacteriprotocol  identifyThe assembled genome was examined using PRISM 1.2.7 , and 12 S. canus have been studied extensively in the past and have revealed a range of compounds with antifungal and bactericidal activities (S. canus ATCC 12647, a known producer of the cardiolipin-targeting antibiotic telomycin. Our analysis has led to the discovery of telomycin’s unique mode of action, as well as the presence of numerous other nonribosomal peptide and polyketide clusters of potentially interesting chemistry and activity within this organism.Different strains of tivities . Here, wLQCG00000000. The version described in this paper is the first version, LQCG01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Specifically, it uses Burrow-Wheeler Transform (BWT) algorithm to directly align bisulfite sequencing reads to a reference genome and implements a novel sliding window based approach with statistical methods for the identification of differentially methylated regions (DMRs). The capability of processing data parallelly allows it to outperform a number of other bisulfite alignment software packages. To demonstrate its utility and performance, we applied it to both real and simulated bisulfite sequencing datasets. The results indicate that Methy-Pipe can accurately estimate methylation densities, identify DMRs and provide a variety of utility programs for downstream methylation data analysis. In summary, Methy-Pipe is a useful pipeline that can process whole genome bisulfite sequencing data in an efficient, accurate, and user-friendly manner. Software and test dataset are available at http://sunlab.lihs.cuhk.edu.hk/methy-pipe/.DNA methylation, one of the most important epigenetic modifications, plays a crucial role in various biological processes. The level of DNA methylation can be measured using whole-genome bisulfite sequencing at single base resolution. However, until now, there is a paucity of publicly available software for carrying out integrated methylation data analysis. In this study, we implemented Methy-Pipe, which not only fulfills the core data analysis requirements (e.g. sequence alignment, differential methylation analysis,  DNA methylation is a biochemical process that predominantly involves the addition of a methyl group to cytosine nucleotides by DNA methyltransferases. This process plays an important role in the regulation of gene expression in both normal and dysfunctional cells etc.) that are posed by different research focuses. Unfortunately, most of the existing tools cannot provide such a comprehensive spectrum of analysis. For example, some software packages are designed for bisulfite sequencing read alignment only Several library preparation protocols have been developed such as Reduced Representation Bisulfite Sequencing (RRBS) (i) BSAligner module, a bisulfite sequencing read alignment module for data pre-processing and sequence alignment that is implemented based on 2BWT http://i.cs.hku.hk/2bwt-tools/downloads/2bwt-lib-v1.0.0-x84-64bit.tar.gz) and SOAP2 http://soap.genomics.org.cn/down/SOAPaligner-v2.20-src.tar.gz); (ii) BSAnalyzer module, a data analysis module implemented to provide a variety of functionalities to facilitate the downstream methylation data analysis. The major functions implemented in this module are: (1) to report the basic statistics and sequencing quality of the data; (2) to calculate the methylation level for any cytosine site and report genome-wide methylation profiles of the analyzed samples; (3) to identify DMRs for paired samples; and (4) to annotate and visualize the methylation data for data mining and easy interpretation. The details of the Methy-Pipe and the implementation of its functional modules will be further elaborated in the following sections.Methy-Pipe is designed to analyze the bisulfite sequencing data from the MethylC-Seq protocol  [3]. TheThe input data for Methy-Pipe consists of high-throughput bisulfite sequencing reads sequenced from either single or paired-end library prepared according to the MethylC-Seq protocol (i) the sequencing adaptors; and (ii) low quality bases  on read ends. The processed reads are then aligned to the insilico converted reference genomes. To prepare the insilico converted reference genomes for methylation awareness alignment, two C depleted reference genomes are built insilico by computationally converting all Cs to Ts in both Watson and Crick strands. Whole genome sequence indices of these two converted genomes are then created using Burrows-Wheeler transform (BWT) algorithm insilico. The pre-processed and converted reads were then aligned to the pre-converted reference genomes. After the alignment, first, we discard those reads that can be aligned back to both the Watson and Crick strands. Then the remaining insilico converted alignable reads are replaced by the original bisulfite sequencing reads and used for downstream methylation data analysis.To align bisulfite sequencing reads back to the reference genome, we implemented BSAligner . BrieflyTo calculate the methylation density, we first count the total number of nucleotide C and T that overlap with each genomic cytosine site across the whole genome. If the sequenced fragment is so short that the sequenced read 1 and 2 overlap each other and the overlapped region covers the genomic cytosine sites, only one sequenced C or T with higher quality score will be counted. Then the MD can be calculated by the following equation:(i) is the total number of sequenced cytosines at the ith cytosine position in the reference genome, suggesting the methylated event, and T(i) is the total number of sequenced thymines at the ith position which is suggestive of unmethylated event. When n equals to 1, MD at a single-base resolution could be calculated.Where, in a given genomic region, n is the total number of cytosines, C(i) determining seed regions; (ii) identifying differentially methylated seed regions; (iii) differentially methylated seed region extension; and (iv) merging of adjacent differentially methylated seed regions. More specifically, to determine seed region, initially, a w-base  sliding window is applied from one end of the chromosomes of two compared samples. A w-base sliding window can be defined as a seed region if it meets the following criteria: (1) for both samples, the sliding window should contain at least m valid CpG sites ; (2) each valid CpG site should be covered by at least n bisulfite sequencing reads . Otherwise this w-base window will be slid downstream with a s-base increment each time  until the aforementioned criteria are satisfied.To identify DMRs genome-wide between two compared samples, a sliding window approach is implemented  with fouNext, to identify differentially methylated seed region, the MD of each valid CpG site is calculated. Mann-Whitney U test is employed to test if the MDs of those valid CpG sites are statistically different  between two compared samples. If the test is statistically significant, this region is identified as a differentially methylated seed region.-base window. If it is also a differentially methylated seed region, the two regions will be merged together. The same procedure will be repeated until the extended region is longer than k bases  or the adjacent region does not satisfy the criteria as a differentially methylated seed region.To extend this region, we use the same approach to interrogate its adjacent downstream w(1) with similar methylation pattern in both samples. For example, both regions are more methylated in one sample than the other or vice versa; (2) with differences in MDs less than 10% in the same sample. Next, all qualified CpG sites within a merged differentially methylated region will be further subjected to χ2 test to assess if the proportion of the sequenced methylated cytosines over the total sequenced methylated and unmethylated cytosines is statistically different between two compared samples . The final merged differentially methylated seed regions with significant difference are considered as putative DMRs.Lastly, we merge the adjacent differentially methylated seed regions within 1000 bases of each other if they share similar methylation profile, i.e. Notably, our algorithm is different from BSmooth http://www.rocksclusters.org).Methy-Pipe is implemented using Perl, R as well as C++. It is designed to run on x86_64 GNU/Linux platform. The data analysis performance can be enhanced by distributing multiple samples to different computing nodes using a Sun Grid Engine (SGE), for example, running on a Rocks cluster . After the alignment, the following output files were created by Methy-Pipe: (i) the aligned reads stored in a text file  with duplicated reads removed ((ii) methylation call data in text files for both Watson and Crick strands. In these files, for each cytosine in the reference genome, the total number of the sequenced methylated cytosines (Cs) and unmethylated cytosines (Ts) as well as the sequence context are reported  reported . Notablyreported .BSAligner, we tested if it can align the bisulfite sequencing reads to the reference genome in an efficient and accurate manner. To this end, we first computationally generated ∼1 million simulated 75 base bisulfite sequencing paired-end reads from the lambda genome. The insert sizes of those simulated paired-end reads ranged from 75 to 600 bases. The methylation level of all the cytosines in CpG dinucleotides are set to be 100%. Using 20 cores on an Intel Xeon 2.80 GHz CPU, it took approximately 25 s to complete the entire alignment, suggesting BSAligner is a very efficient alignment tool. The resultant alignment accuracy is 99.9%, with a mappability of 99.9%, suggesting an excellent performance.To further evaluate the performance of Next, to test the accuracy of Methy-Pipe alignment for the bisulfite sequencing reads with different levels of methylations, we obtained a simulated dataset from human genome that contains ∼1 million bisulfite sequencing paired-end reads with the simulated methylation level ranging from as low as 5% to 100%. By aligning these reads using BSAligner, Methy-Pipe can accurately detect the simulated methylation states of cytosines in any sequence context while the mapping efficiency is completely unaffected .insilico simulated methylation data, BSAligner outperformed Bismark in terms of the computation time  is the first step towards the quantification of methylation data. To access if MD, calculated by Methy-Pipe based on the whole genome bisulfite sequencing data, can represent the methylation state of each CpG site accurately, we compared it with the methylation state measured by another independent platform, Illumina Infinium HumanMethylation450 BeadChip As a result, there is a good concordance between the MDs obtained from Methy-Pipe and the values obtained from Illumina Infinium HumanMethylation450 array for CpG sites analyzed. The Pearson correlation coefficient of 0.94 for placenta sample  indicate(i) a plot of base compositions of four nucleotides at each sequencing cycle, in which a high percentage of T and low percentage of C are expected due to the base conversion involved in the bisulfite sequencing procedure (etc.); (ii) a plot of the distribution of the insert size for the paired-end sequencing. This can help to monitor if the insert size of the sequencing library is as expected or not , and bisulfite conversion rate estimated by the spiked lambda genome if available . In addirocedure . A deviad or not .(i) to generate genome-wide methylation profiles using MDs of fixed windows across the whole genome to visualize the MDs in a scatter plot. In the scatter plot, each dot represents a genomic region with a fixed length (i.e. 100 kb). The MDs of these fixed windows are plotted against their genomic locations in the reference genome ((ii) to provide MDs for cytosines under different sequence contexts   . When ap C or T) . Further C or T) , suggestetc.) were found to be hypo-methylated in a genome-wide manner in various cancers In addition to investigating the methylation states at whole genome level, studying the methylation profiles around specific genomic regions can also shed light on how DNA methylation affects gene expression. For example, MDs around transcription start sites (TSSs) are commonly correlated with the expression levels of epigenetically regulated genes (i) A DMR list file reporting the detailed information of the identified DMRs, such as genomic locations, methylation states (hypomethylated or hypermethylated), cytosine and thymine counts, and the p-values for the statistical test, etc. ((ii) A DMR annotation file providing the information on the neighboring gene(s) for each DMR . To demonstrate how to use this algorithm in Methy-Pipe for the identification of DMRs, we applied it to the placenta and buffy coat datasets. As a result, two files were created as the outputs of the identification of DMRs: st, etc. ; (ii) A each DMR .(i) the values was first calculated as the mean value of a group of the probes on HumanMethylation450 array within 500 bp window; (ii) the hypermethylated regions were defined by the difference of mean values between placenta and buffy coat larger than 25%; (iii) the hypomethylated regions were defined by the difference of mean values between the two samples larger than 25% in a reverse direction; (iv) The unchanged regions were defined by the difference of mean values between the two samples within 3%. Based on the above definition, 676, 2,650 and 9,249 regions were identified as hypermethylated, hypomethylated and unchanged regions. When comparing the above regions with the corresponding regions identified from Methy-Pipe using default parameter settings  from data with comparatively low sequencing depth , we could achieve 97%, 91%, and 96% accuracy for the detection of hypermethylated, hypomethylated, and unchanged regions . As a result, 1,688 hypermethylated and 6,793 hypomethylated DMRs were associated with the known annotated genes. For example, the promoter of RASSF1A gene is more hypermethylated in placenta compared with maternal buffy coat , which iTo further demonstrate the computational cost of Methy-Pipe, we also tested it on 10 million 75 bp paired-end bisulfite sequencing reads. It took 33 minutes to complete the whole analysis with peak memory usage of 25 GB based on an Intel Xeon X5675 CPU using 20 cores.In this study, we designed and implemented Methy-Pipe, an integrated whole genome bisulfite sequencing data analysis pipeline. It not only fulfills the core data analysis requirements such as bisulfite-treated sequencing read alignment, methylation level inference, and DMR identification but also provides a variety of utility programs to further annotate and visualize the resulting methylation data. Using real datasets from human placenta and maternal buffy coat samples, we demonstrated that Methy-Pipe can efficiently and accurately analyze the whole genome bisulfite sequencing data. Thus, this new pipeline would facilitate us to develop next generation sequencing based diagnostic approaches based on the DNA methylation marker in many research areas of medical genomics such as prenatal diagnosis When compared with many previously reported whole genome bisulfite sequencing data analysis software packages, Methy-Pipe appears to demonstrate more functionality and is easier to use. First, it integrates the core and the downstream data analysis modules into one package so that the end user can explore the biological significance of methylation. In addition, Methy-Pipe can take advantage of the high-performance computing clusters by utilizing SGE to parallelize data analyzes, which could dramatically speed up the analysis of bisulfite sequencing data that is normally huge and demands intensive computing power.Our BSAligner allows efficient alignment of sequencing reads. Compared to the majority of aligners designed for bisulfite sequencing data alignment, its performance has been greatly improved by integrating quality control filters before the read alignment is carried out. First, low quality bases of the two ends of the sequenced reads can be filtered, which decreases methylation call errors. Second, sequence adaptors can also be filtered to reduce the adaptor contamination for the short reads during the methylation inference. In addition, BSAligner adopts a methylation unbiased approach, in which any available cytosine in the sequenced read after bisulfite treatment and all cytosines in the reference genome are converted into thymines before the alignment. In the BSAligner, it can directly map the converted bisulfite reads to converted reference genome using the BWT algorithm, thus eliminating the time consuming step of converting all Cs to Ts during the alignment. As a result, the performance of BSAligner is better than that of Bismark Table S1The example output of BSAligner.(DOCX)Click here for additional data file.Table S2The example output of methylation call by Methy-Pipe.(DOCX)Click here for additional data file.Table S3The example output of basic statistics of methylation data reported by Methy-Pipe.(DOCX)Click here for additional data file.Table S4The performance comparison between BSAligner and Bismark.(DOCX)Click here for additional data file.Table S5The example output of methylation densities for the regions of interest.(DOCX)Click here for additional data file.Table S6The example list of identified DMRs.(DOCX)Click here for additional data file.Table S7The example list of DMRs annotated with the closest genes.(DOCX)Click here for additional data file.