Over the last year, NISC has put into production two additional NextGen DNA sequencing machines, an Illumina HiSeq2000 and a Roche GS454, making our current suite of production NextGen sequencing machines total 4 HiSeq2000s, 2 GAiiXs, 3 MiSeqs and 2 GS454s. Using these platforms, we have generated about 400 billion reads in the past year alone. Though we remain consistently at a level of a mid-scale genome sequencing center, we have maintained advantageous economies of scale while remaining relatively agile. While NISC has undertaken projects of many sizes and types throughout the years, from ESTs to SAGE sequencing, the NISC Comparative Sequencing Program has been the most productive over-arching success, beginning with the sequencing of mouse BACs orthologous to human chromosome 7 at the start of the mouse genome project and extending to over 75 species across numerous targets, including the flagship CFTR target that encompasses 1 MB of human chromosome 7. This BAC-based sequencing approach found great utility in scouting new genomes and for specialized targeting of complex genomic regions containing duplications and structural rearrangements that made them intractable by traditional genomic sequencing approaches. There were 3 BAC related publications this reporting period (5, 10, 16). In keeping with the Comparative Sequencing interests, several years ago NISC implemented an amplicon-based Sanger sequencing pipeline designed to focus on intra-species variation. Numerous clinically relevant projects were designed to amplify and sequence specific genes and regions of interest in small groups of human subjects, yielding great insights into disease related genotype/phenotype combinations. The flagship ClinSeq Project greatly advanced the study of atherosclerosis by providing sequence data for 250 genes in over 500 volunteers. While this approach was extremely productive, the combination of large volumes of high quality sequence data generated by the Illumina platform, along with efficient whole exome genomic enrichment techniques evaluated and adopted by NISC has allowed us to transition to an even more cost-effective approach that provides an increasingly comprehensive data set. As a consequence of these advances, NISC no longer offers Sanger-based amplicon targeted sequencing in production mode. Four publications related to those previous efforts are listed in the publications section of this report (2, 12, 17, 23). The adoption of many new sequencing protocols in production created the commensurate need for dramatic changes to sample tracking, flow control and primary analysis pipelines. Rapid design, development and implementation of new Laboratory Information Management System (LIMS) by a dedicated team has met the initial challenges and continues to evolve quickly to adapt to a continuous flow of changes. A combination of talented IT staff and bioinformaticians have met the challenges of extremely large and complex data sets by implementing and continuously adapting pipeline programs to support rapidly evolving software associated with each of the sequencing platforms. Beyond primary analysis that results in DNA basecalls and quality scores, NISC has worked closely with members of other NHGRI research groups to implement and support high-throughput production of biologically relevant secondary analysis. One shining example of these efforts is the production scale processing of Whole Exome Sequencing (WES) data to all of our clients, the end product of which is distilled sets of variants of interest that are accessible in user-friendly fashion by the use of the in-house developed VarSifter program. The success of these programs has led to an increasing number of projects from a growing number of investigators. The implementation of improved project management tools is helping to address the challenges associated with such growth, which is now yielding results as publications for WES (n = 6) (7, 13, 15, 20, 21, 24), miRNA (n = 1) (11), Whole Genome Sequencing, Assembly and/or Annotation (n = 4) (4, 14, 18, 25), custom targeted sequencing (n = 3) (1, 6, 22), RNAseq (n = 3) (1, 8, 18), microbiome studies (n = 3) (3, 19, 25), and HIV antibody studies (n=4) (9, 26, 27, 28). In the foreseeable future, NISC is well positioned to provide next-gen sequence data for several large, multi-year projects, for example, the Skin Microbiome Project, and Mouse Methylome Project, a collaboration with NIEHS, as well as expanding the access to sequencing by Intramural NHGRI investigators through a new internal review process for advancing the most promising projects. Our focus is to increase operational efficiencies of the next-gen pipeline, refine existing protocols, implement additional protocols as new sample/experimental types are requested from researchers and continue to expand the value added data analysis packages available. We are currently testing specific applications for new technologies including the Ion Torrent sequencing instrument, PacBio generated data as a foundation for microbial genome sequencing, and the OpGen/Argus physical restriction mapping platform. Furthermore, we will continue to monitor developments in the rapidly evolving sequencing and informatics technologies, implementing those we deem most appropriate for the sequence data we produce for collaborating investigators.

Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2013
Total Cost
$9,119,432
Indirect Cost
Name
National Human Genome Research Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Jo, Jay-Hyun; Deming, Clay; Kennedy, Elizabeth A et al. (2016) Diverse Human Skin Fungal Communities in Children Converge in Adulthood. J Invest Dermatol :
Sood, R; Hansen, N F; Donovan, F X et al. (2016) Somatic mutational landscape of AML with inv(16) or t(8;21) identifies patterns of clonal evolution in relapse leukemia. Leukemia 30:501-4
Beck, Tyler F; Mullikin, James C; NISC Comparative Sequencing Program et al. (2016) Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants. Clin Chem 62:647-54
Boyden, Steven E; Desai, Avanti; Cruse, Glenn et al. (2016) Vibratory Urticaria Associated with a Missense Variant in ADGRE2. N Engl J Med 374:656-63
Conlan, Sean; Park, Morgan; Deming, Clayton et al. (2016) Plasmid Dynamics in KPC-Positive Klebsiella pneumoniae during Long-Term Patient Colonization. MBio 7:
Justice, Cristina M; Bishop, Kevin; Carrington, Blake et al. (2016) Evaluation of IRX Genes and Conserved Noncoding Elements in a Region on 5p13.3 Linked to Families with Familial Idiopathic Scoliosis and Kyphosis. G3 (Bethesda) 6:1707-12
Soto, Cinque; Ofek, Gilad; Joyce, M Gordon et al. (2016) Developmental Pathway of the MPER-Directed HIV-1-Neutralizing Antibody 10E8. PLoS One 11:e0157409
Ng, David; Hong, Celine S; Singh, Larry N et al. (2016) Assessing the capability of massively parallel sequencing for opportunistic pharmacogenetic screening. Genet Med :
Tsai, Yu-Chih; Conlan, Sean; Deming, Clayton et al. (2016) Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing. MBio 7:e01948-15
Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang et al. (2016) Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody. Cell 165:449-63

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