Next Generation Sequencing Software Research Articles

Comprehensive comparison of cloud-based NGS data analysis and alignment tools

Abstract Next-Generation Sequencing (NGS) is very helpful for conducting DeoxyriboNucleic Acid (DNA) Sequencing. DNA sequencing is the process for determining the order (sequence) of the main chemical bases in the DNA. Analyzing human DNA sequencing is important for determining the possibility that a person will develop certain diseases, and/or the ability to respond to medication. However, the NGS process is a complicated and resource-hungry technical process. To solve this dilemma, the majority of NGS software systems are deployed as cloud-based services distributed over cloud-based platforms. Cloud-based platforms provide promising solutions for the computationally intensive tasks required by the NGS data analysis. This work provides a comprehensive investigation of cloud-based NGS data analysis and alignment tools, both the commercial and the open-source tools. We also discuss in detail the main features and setup requirements for each tool, and then compare and contrast between them. Moreover, we extensively analyze and classify the studied NGS data analysis and alignment tools to help NGS biomedical researchers and clinicians in finding appropriate tools for their work, while understanding the similarities and the differences between them.

Evaluation of Commercial Next-Generation Sequencing Bioinformatics Software Solutions

Next-generation sequencing (NGS) diagnostics continue to expand rapidly in clinical medicine. An ever-expanding menu of molecular biomarkers is deemed important for diagnostic, prognostic, and therapeutic assessment in patients. The increasing role of NGS in the clinic is driven mainly by the falling costs of sequencing. However, the data-intensive nature of NGS makes bioinformatic analysis a major challenge to many clinical laboratories. Critically needed NGS bioinformatics personnel are hard to recruit and retain in small- to mid-size clinical laboratories. Also, NGS software often lacks the scalability necessary for expanded clinical laboratory testing volumes. Commercial software solutions aim to bridge the bioinformatics barrier via turnkey informatics solutions tailored specifically for the clinical workplace. Yet, there has been no systematic assessment of these software solutions thus far. This article presents an end-to-end vendor evaluation experience of commercial NGS bioinformatics solutions. Six different commercial vendor solutions were assessed systematically. Key metrics of NGS software evaluation to aid in the robust assessment of software solutions are described. Comprehensive feedback, provided by the TriCore Reference Laboratories molecular pathology team, enabled the final vendor selection. Many key lessons were learned during the software evaluation process, which are described herein. This article aims to provide a detailed road map for small- to mid-size clinical laboratories interested in evaluating commercial bioinformatics solutions available in the marketplace.

Multi-gene testing in neurological disorders showed an improved diagnostic yield: data from over 1000 Indian patients.

Neurological disorders are clinically heterogeneous group of disorders and are major causes of disability and death. Several of these disorders are caused due to genetic aberration. A precise and confirmatory diagnosis in the patients in a timely manner is essential for appropriate therapeutic and management strategies. Due to the complexity of the clinical presentations across various neurological disorders, arriving at an accurate diagnosis remains a challenge. We sequenced 1012 unrelated patients from India with suspected neurological disorders, using TruSight One panel. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. We were able to detect mutations in 197 genes in 405 (40%) cases and 178 mutations were novel. The highest diagnostic rate was observed among patients with muscular dystrophy (64%) followed by leukodystrophy and ataxia (43%, each). In our cohort, 26% of the patients who received definitive diagnosis were primarily referred with complex neurological phenotypes with no suggestive diagnosis. In terms of mutations types, 62.8% were truncating and in addition, 13.4% were structural variants, which are also likely to cause loss of function. In our study, we observed an improved performance of multi-gene panel testing, with an overall diagnostic yield of 40%. Furthermore, we show that NGS (next-generation sequencing)-based testing is comprehensive and can detect all types of variants including structural variants. It can be considered as a single-platform genetic test for neurological disorders that can provide a swift and definitive diagnosis in a cost-effective manner.

P001 Validation of ALLType™ next-generation sequencing in Brazilian samples

Aim Validation of HLA typing by next-generation sequencing (NGS) demands evidence of the assay’s accuracy compared to Sanger sequencing. We evaluated the AllType™ NGS (One Lambda) and TypeStreamVisual™NGS analysis software (One Lambda) for accuracy on HLA genotyping. Methods 48 Brazilian DNA samples selected for allelic diversity were sequenced for HLA class I and class II genes following AllType™ NGS protocol using automation of the Ion Chef and S5 sequencing system (One Lambda). Sequencing data was analyzed by TypeStreamVisual™ software 1.0.0 with IMGTHLA database 3.27.0.8. All samples were previously typed for HLA-A, B, C, DRB1, DQB1 and DPB1 by SeCore Sequence-Based Typing kits (One Lambda) using uTYPE HLA sequence analysis software (One Lambda).Evaluation was done comparing NGS results with previously assigned by Sanger’s method. Results AllTypeTM NGS and SeCore results were 100% concordant for HLA- A, B, C, DRB1, DQB1 and DPB1 loci. Following ambiguities were detected for class I and class II:B*15:01:01 B*15:03:01/B*15:39:01 B*15:54; B*07:02:01 B*08:01:01/B*08:156 B*42:01:01; DRB1*12:01:01 DRB1*13:02:01/DRB1*12:10 DRB1*13:02:01; DQB1*06:03:01 DQB1*06:09:01/DQB1*06:41 DQB1*06:88; DPB1*04:01:01 DPB1*04:02:01/DPB1*105:01 DPB1*126:01 Allelic imbalance cases occurred most in DQB1 locus, all involving DQB1*02. All null alleles required identification by the National Marrow Donor Program could be ruled out as potential results. Reproducibility was validated by 3 samples tested 3 times in the same assay run and 4 samples tested in 3 different runs. 20 blind samples sequenced also by NGS were tested and approved up to 3-field result for all loci. Conclusions We concluded that AllType™ NGS is reliable to be introduced in clinical routine for HLA-A, B, C, DRB1, DQB1 and DPB1 loci. Although HLA-B ambiguities needed additional tests, there was enormous reduction in the number of ambiguities detected by AllType™ providing highest cost benefit than Sanger sequencing. Download : Download high-res image (548KB) Download : Download full-size image

Applications of Probe Capture Enrichment Next Generation Sequencing for Whole Mitochondrial Genome and 426 Nuclear SNPs for Forensically Challenging Samples.

The application of next generation sequencing (NGS) for the analysis of mitochondrial (mt) DNA, short tandem repeats (STRs), and single nucleotide polymorphism (SNPs) has demonstrated great promise for challenging forensic specimens, such as degraded, limited, and mixed samples. Target enrichment using probe capture rather than PCR amplification offers advantages for analysis of degraded DNA since two intact PCR primer sites in the template DNA molecule are not required. Furthermore, NGS software programs can help remove PCR duplicates to determine initial template copy numbers of a shotgun library. Moreover, the same shotgun library prepared from a limited DNA source can be enriched for mtDNA as well as nuclear markers by hybrid capture with the relevant probe panels. Here, we demonstrate the use of this strategy in the analysis of limited and mock degraded samples using our custom probe capture panels for massively parallel sequencing of the whole mtgenome and 426 SNP markers. We also applied the mtgenome capture panel in a mixed sample and analyzed using both phylogenetic and variant frequency based bioinformatics tools to resolve the minor and major contributors. Finally, the results obtained on individual telogen hairs demonstrate the potential of probe capture NGS analysis for both mtDNA and nuclear SNPs for challenging forensic specimens.

Collection and storage of HLA NGS genotyping data for the 17th International HLA and Immunogenetics Workshop

For over 50 years, the International HLA and Immunogenetics Workshops (IHIW) have advanced the fields of histocompatibility and immunogenetics (H&I) via community sharing of technology, experience and reagents, and the establishment of ongoing collaborative projects. Held in the fall of 2017, the 17th IHIW focused on the application of next generation sequencing (NGS) technologies for clinical and research goals in the H&I fields. NGS technologies have the potential to allow dramatic insights and advances in these fields, but the scope and sheer quantity of data associated with NGS raise challenges for their analysis, collection, exchange and storage. The 17th IHIW adopted a centralized approach to these issues, and we developed the tools, services and systems to create an effective system for capturing and managing these NGS data. We worked with NGS platform and software developers to define a set of distinct but equivalent NGS typing reports that record NGS data in a uniform fashion. The 17th IHIW database applied our standards, tools and services to collect, validate and store those structured, multi-platform data in an automated fashion. We have created community resources to enable exploration of the vast store of curated sequence and allele-name data in the IPD-IMGT/HLA Database, with the goal of creating a long-term community resource that integrates these curated data with new NGS sequence and polymorphism data, for advanced analyses and applications.

P021 Managing NGS HLA genotyping data in the 17th workshop database

Aim The 17th International HLA and Immunogenetics Workshops (IHIW) focuses on the application of next generation sequencing (NGS) technologies for histocompatibility and immunogenomic clinical and research goals. However, the scope and sheer quantity of NGS data raises challenges for their analysis, collection, exchange and storage. The 17th IHIW has adopted a centralized approach to these issues, with the goals of developing tools, services and systems to capture and manage these NGS data, and leveraging the vast store of sequence and allele data in the IPD-IMGT/HLA Database. Methods We worked with NGS platform and software developers to define NGS typing reports that record NGS data in a uniform fashion. The IHIW database and data-storage facility houses typing reports, FASTQ files, subject and specimen data, and pedigrees, and manages the accounts and data-access privileges for 17th IHIW participants. IHIW database tools and services are scripted in the Perl, R or Python languages. Results We defined three distinct typing report formats [Histoimmunogenetics Markup Language (HML), IHIW eXtensible Markup Language (XML), and GenDx XML formats] that record HLA genotyping data and meta-data in equivalent fashion. Our tools convert HML and GenDx XML to IHIW XML, automate and streamline the submission of IHIW XML reports, validate each typing report upon submission, and automate the standardized reporting of novel polymorphisms. All 17th IHIW HLA data are standardized to IPD-IMGT/HLA Database version 3.25.0, with genotype data generated under other database versions converted to 3.25.0. Conclusions The 17th IHIW database applies our standards, tools and services to consume, validate and store structured NGS HLA data generated by GenDx, Illumina, Immucor, Omixon and One Lambda in an automated fashion. The HLA genotyping data and meta-data in these typing reports are validated and standardized for use in 17th IHIW projects, and the database itself will be available for future workshops.

P252 Conceptualization to implementation in 90 days of full gene HLA typing using mia fora HLA flex kits at Jeevan stem cell foundation, India

Aim Next Generation Sequencing (NGS) is rapidly becoming the method of choice for HLA typing for Hematopoietic Stem Cell Transplantation. Jeevan Stem Cell Foundation, Chennai, India validated the full gene HLA typing using Immucor’s MIA FORA FLEX kits from conceptualization to implementation in ninety days. This implementation, in line with the Indian Government’s policy of “Make in India”, enabled Jeevan to establish a cost effective high resolution HLA typing to international standards in India to support the transplant physicians and registries in the region and prevent Indian DNA being shipped to other countries. Methods Reference DNA used for validation were previously typed by SBT included samples from UK NEQAS, FRED HUTCHINSON and Jeevan’s registry samples. DNA was extracted using the Qiacube. HLA typing for A, B, C, DRB1 and DQB1 were performed. All workflows were performed according to the supplier’s instructions. NGS sequencing was done using the mid output flow cell kit from Illumina and sequenced on the Illumina MiniSeq platform. Immucor’s proprietary software was used to analyse the data. Results Results were evaluated per allele on the 2nd field consensus compared to the reference, the number of allelic ambiguities, genotype ambiguities and allelic drop outs were measured. The second field resolution was used because higher resolution typing was not available for all samples. MIA FORA NGS software provided high resolution typing for all samples. There results from NGS typing compared to SBT typing are shown in Table 1. Conclusions The implementation of the MIA FORA FLEX HLA typing kit at Jeevan is the first outside USA. With the MIA FORA kits, the turnaround time will depend upon the number of samples. In our experience reports for 48 samples can be delivered within 72 h. There were no ambiguous results and no allelic dropout. The proprietary software of Immucor was easy to use and significantly reduced the analysis time. Download high-res image (213KB) Download full-size image

P066 Next-generation sequencing (NGS) HLA typing: Beyond allele assignment

Aim To expand the analytical potential of NGS in HLA typing. Studies based on SNP analysis have revealed the DNA sequence of HLA genes to carry important biological information typically overlooked in the simple exercise of allele assignment. NGS software is optimised to match sample sequences against databases of HLA allele sequence references. The goal here, however, is to create a method to open the data collected by NGS to any kind of query. Methods BLAST is still one of the most robust and efficient sequence-matching and sequence-alignment methods. Typically BAST databases are built with reference sequences against which a sample sequence is queried for matches. Here we reverse the approach and build a database of sample sequences against which we query for matches for particular sequences of interest following these steps: 1. Compile a database of sample sequences included in a given study. 2. Convert sample sequences to FASTA format. 3. Create a BLAST database with the study sample sequences. 4. Run any BLAST query against the BLAST database of the study sample sequences. Results This method was implemented using a dataset collected for typing purposes. The HLA-C 3’UTR polymorphisms which bind to miRNAs and regulate the level of expression (Nature 2011; 472:7344) were used. The BLAST output provided accurate information about which sequences carried the query polymorphism, which matched what is known about the association of these SNPs with HLA-C alleles. This method has also been successfully used to look for mutations that render HLA alleles null, and other SNP analysis queries. Conclusions NGS provides data that goes beyond the need for simple allele assignment. The method presented here provides (1) a robust and reliable way to store this accumulated information, (2) a quick and simple way to query this database of sequence data; and (3) an open method to ask any sequence question. M. Li: Employee; Company/Organization; Immucor.

Abstract C04: Comparative analysis of bioinformatic tools for the detection of viral DNA sequences in tumor cells

Abstract Cancer health disparities exist among different ethnicities and races. Various factors such as lifestyle, environmental exposure, genetics, and epigenetics are thought to play a role in the existence of these disparities. Viruses, which outnumber cells in the human body by 100-fold, have a major impact on human health. Viruses have been estimated to cause 15-20% of human cancers, and we expect that some of these oncogenic viruses may have the potential to impact health disparities. Next generation sequencing (NGS) technologies are being used to investigate novel virus-cancer associations and interactions, and several bioinformatics tools for the detection and analysis of virus sequences in human NGS data have recently become available. These tools have not been validated, however, for use in human cancers with extremely low levels of viral sequences. In this study, we have compared the sensitivity and specificity of READSCAN to a manually constructed analysis workflow in a common, commercial NGS web application. READSCAN is a freely available software application that utilizes automated “digital subtraction” to eliminate host reads and identify specific virus sequences in NGS data. The functionality of this tool was compared to a workflow constructed within the web interface for the commercially available NGS software Partek. This constructed workflow first subtracted human sequences and then aligned the remaining sequences against select viral genomes. The sensitivity of the various tools was compared using NGS data obtained from human cytomegalovirus (HCMV) infected cells. Specificity of the tools was determined by analyzing the same data set against the Epstein-Barr virus (EBV) and Human Papilloma virus type 16 (HPV-16). Partek and READSCAN detected 69.66% and 60.54% of the input number of reads as HCMV sequences. Under these conditions, neither bioinformatics tool detected EBV or HPV16 specific reads in the HCMV infected cell data. This indicated that both Partek and READSCAN were capable of readily detecting the presence of large amounts of virus reads in NGS data from infected cells in a specific manner. In order to test the ability of these tools to detect viral specific reads in NGS data from tumors, data obtained from the HPV16 positive cervical squamous cell carcinoma cell line SiHa were analyzed. Both Partek and READSCAN detected six reads out of 969,798 total reads as HPV16 sequences; EBV or HCMV sequences were not detected. Our results are in agreement with previously published observations for this SiHa cell line, in which five HPV16 specific reads were detected. While we are still in the process of testing additional bioinformatics tools and configurations, our results attest that the open–source bioinformatics tool READSCAN and the commercially available Partek are comparable in performance, but differ in utility. Partek is faster, has a user-friendly interface, and a knowledge of Linux commands is not required. READSCAN lacks these features, but it is free and has an algorithm more clearly proscribed in the literature for digital subtraction. Next steps include utilizing a wider range of digital subtraction software, evaluating a broader range of NGS data from demographically diverse origins, and investigating discordance as it is found between alternative tools and configuration parameters. Ultimately, an ensemble of tools and configuration contexts is expected to yield critical information on the role of viruses in cancer and associated health disparities Citation Format: Barbara Swanson, Scott Harrison, Dukka KC, Perpetua Muganda. Comparative analysis of bioinformatic tools for the detection of viral DNA sequences in tumor cells. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C04. doi:10.1158/1538-7755.DISP13-C04

OR37: INCREASED LEVELS OF CELL-FREE CIRCULATING DONOR DNA IN RECIPIENT DETECTED BY NEXT GENERATION SEQUENCING OF HLA AMPLICONS: A POSSIBLE INDICATOR OF KIDNEY GRAFT REJECTION

Aim The circulation of kidney transplant recipients has been reported to contain DNA from the graft, associated with rejection. We previously reported the development of a sensitive, accurate and precise method to quantitatively analyze contrived mixtures of two cell-line DNAs by next generation sequencing (NGS) of short HLA amplicons. Here we report the application of this method to analyze the cell-free circulating (cfc) DNA from kidney transplant recipients that fell into one of four categories at that time of sample collection: (1) successful transplant (Tx), (2) experiencing acute rejection (AR), (3) experiencing chronic rejection (CR), or (4) experiencing acute dysfunction (ADNR) but no rejection. Methods DNA from recipient plasma was amplified using primers that targeted a short region of HLA-DPB1 or DQB1 exon 2 (⩽150 bp to amplify small DNA fragments in plasma). Primers included Illumina Miseq tags. A second round of PCR was performed to introduce Miseq indices (to identify individual samples) and sequencing adapters. Amplicons were sequenced on a 2 × 150 output run to obtain sequence in both directions and analyzed with a modified version of Conexio Assign ATF 454 software as well as SWISSA, an in-house NGS software. Both softwares allowed for rapid digital analysis of HLA alleles present from graft recipients and donors. Results Donor HLA alleles in recipient plasma were readily identified and separated from background “noise” i.e., sequences generated by PCR or sequencing errors. The average number of sequence reads when processing with SWISSA was 146,000 per sample. Using cell-line mixtures, with an input of 8 ng of DNA, we detected alleles down to 0.25% (the lowest amount we tested). In a study of 29 patients, the average percentages of donor DNA were 2.1 ± 2.3, 1.3 ± 1.4, 0.5 ± 0.4 and 0.2 ± 0.3 for patients who had AR, CR, ADNR or Tx, respectively. Conclusion Illumina MiSeq sequencing can be used to noninvasively detect and quantify the presence of donor DNA in kidney transplant recipients by analysis of plasma. The average percentage of cfc donor DNA correlated with an increasing severity of rejection in this initial exploratory study.

Harnessing virtual machines to simplify next-generation DNA sequencing analysis

The growth of next-generation sequencing (NGS) has not only dramatically accelerated the pace of research in the field of genomics, but it has also opened the door to personalized medicine and diagnostics. The resulting flood of data has led to the rapid development of large numbers of bioinformatic tools for data analysis, creating a challenging situation for researchers when choosing and configuring a variety of software for their analysis, and for other researchers trying to replicate their analysis. As NGS technology continues to expand from the research environment into clinical laboratories, the challenges associated with data analysis have the potential to slow the adoption of this technology. Here we discuss the potential of virtual machines (VMs) to be used as a method for sharing entire installations of NGS software (bioinformatic 'pipelines'). VMs are created by programs designed to allow multiple operating systems to co-exist on a single physical machine, and they can be made following the object-oriented paradigm of encapsulating data and methods together. This allows NGS data to be distributed within a VM, along with the pre-configured software for its analysis. Although VMs have historically suffered from poor performance relative to native operating systems, we present benchmarking results demonstrating that this reduced performance can now be minimized. We further discuss the many potential benefits of VMs as a solution for NGS analysis and describe several published examples. Lastly, we consider the benefits of VMs in facilitating the introduction of NGS technology into the clinical environment. brian.wilhelm@umontreal.ca.

From next-generation sequencing alignments to accurate comparison and validation of single-nucleotide variants: the pibase software

Scientists working with single-nucleotide variants (SNVs), inferred by next-generation sequencing software, often need further information regarding true variants, artifacts and sequence coverage gaps. In clinical diagnostics, e.g. SNVs must usually be validated by visual inspection or several independent SNV-callers. We here demonstrate that 0.5–60% of relevant SNVs might not be detected due to coverage gaps, or might be misidentified. Even low error rates can overwhelm the true biological signal, especially in clinical diagnostics, in research comparing healthy with affected cells, in archaeogenetic dating or in forensics. For these reasons, we have developed a package called pibase, which is applicable to diploid and haploid genome, exome or targeted enrichment data. pibase extracts details on nucleotides from alignment files at user-specified coordinates and identifies reproducible genotypes, if present. In test cases pibase identifies genotypes at 99.98% specificity, 10-fold better than other tools. pibase also provides pair-wise comparisons between healthy and affected cells using nucleotide signals (10-fold more accurately than a genotype-based approach, as we show in our case study of monozygotic twins). This comparison tool also solves the problem of detecting allelic imbalance within heterozygous SNVs in copy number variation loci, or in heterogeneous tumor sequences.

NGS catalog: A database of next generation sequencing studies in humans

Next generation sequencing (NGS) technologies have been rapidly applied in biomedical and biological research since its advent only a few years ago, and they are expected to advance at an unprecedented pace in the following years. To provide the research community with a comprehensive NGS resource, we have developed the database Next Generation Sequencing Catalog (NGS Catalog, http://bioinfo.mc.vanderbilt.edu/NGS/index.html), a continually updated database that collects, curates and manages available human NGS data obtained from published literature. NGS Catalog deposits publication information of NGS studies and their mutation characteristics (SNVs, small insertions/deletions, copy number variations, and structural variants), as well as mutated genes and gene fusions detected by NGS. Other functions include user data upload, NGS general analysis pipelines, and NGS software. NGS Catalog is particularly useful for investigators who are new to NGS but would like to take advantage of these powerful technologies for their own research. Finally, based on the data deposited in NGS Catalog, we summarized features and findings from whole exome sequencing, whole genome sequencing, and transcriptome sequencing studies for human diseases or traits.

Retraction Note: Evaluation of next-generation sequencing software in mapping and assembly

The publisher is retracting this Review. The same Review was made available online on 28 April 2011 and published in the June issue of Journal of Human Genetics (doi:10.1038/jhg.2011.43). The Journal of Human Genetics and the publisher would like to apologize to the readers of the journal for the inconvenience caused by this inadvertent error.

Evaluation of next-generation sequencing software in mapping and assembly

Next-generation high-throughput DNA sequencing technologies have advanced progressively in sequence-based genomic research and novel biological applications with the promise of sequencing DNA at unprecedented speed. These new non-Sanger-based technologies feature several advantages, when compared with traditional sequencing methods in terms of higher sequencing speed, lower per run cost and higher accuracy. However, reads from next-generation sequencing (NGS) platforms, such as 454/Roche, ABI/SOLiD and Illumina/Solexa, are usually short, thereby restricting the applications of NGS platforms in genome assembly and annotation. We presented an overview of the challenges that these novel technologies meet and particularly illustrated various bioinformatics attempts on mapping and assembly for problem solving. We then compared the performance of several programs in these two fields and further provided advices on selecting suitable tools for specific biological applications.Journal of Human Genetics advance online publication, 16 June 2011; doi:10.1038/jhg.2011.62.

Evaluation of next-generation sequencing software in mapping and assembly

Next-generation high-throughput DNA sequencing technologies have advanced progressively in sequence-based genomic research and novel biological applications with the promise of sequencing DNA at unprecedented speed. These new non-Sanger-based technologies feature several advantages when compared with traditional sequencing methods in terms of higher sequencing speed, lower per run cost and higher accuracy. However, reads from next-generation sequencing (NGS) platforms, such as 454/Roche, ABI/SOLiD and Illumina/Solexa, are usually short, thereby restricting the applications of NGS platforms in genome assembly and annotation. We presented an overview of the challenges that these novel technologies meet and particularly illustrated various bioinformatics attempts on mapping and assembly for problem solving. We then compared the performance of several programs in these two fields, and further provided advices on selecting suitable tools for specific biological applications.

A high-throughput-sequence analysis infrastructure technology investigation framework for the evaluation of next-generation sequencing software

High-throughput sequencing (HTS) is an emerging technology that promises to deliver unparalleled information on genomic variations. As technology evolves and matures, and as a deeper understanding of this technology is gained, new and upgraded tools for analyzing HTS will become available and will need to be evaluated and validated. To facilitate this cumbersome task, we have developed an HTS validation framework into which both in-house-generated synthetic datasets and well-characterized experimental datasets have been incorporated for controlled testing and evaluation of these analysis tools. Currently, the framework can be used to assess algorithms for short-read mapping, variant calling and RNA-Seq-derived gene expression measurements. The framework is deployed in the Amazon EC2 cloud so that it is available to the broader research community. Using our framework, researchers can further validate interfaced applications with preferred parameters, upload their own datasets for processing, and interface new applications with the framework for validation and comparison. We report the performance of several alignment, variant calling and RNA-Seq analytic tools that have been tested with our framework. We also provide feedback on the challenges and benefits of Amazon EC2 deployment.

A high-throughput-sequence analysis infrastructure technology investigation framework for the evaluation of next-generation sequencing software

A high-throughput-sequence analysis infrastructure technology investigation framework for the evaluation of next-generation sequencing software