Aug2014 abrf interlaboratory study plans

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ABRF update

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  • ENCyclopedia Of DNA Elements
    The National Human Genome Research Institute (NHGRI) launched a public research consortium named ENCODE, the Encyclopedia Of DNA Elements, in September 2003, to carry out a project to identify all functional elements in the human genome sequence. The project started with two components - a pilot phase and a technology development phase.
  • Aug2014 abrf interlaboratory study plans

    1. 1. The ABRF Next Generation Sequencing Study: Multi-Platform and Cross-Methodological Reproducibility of RNA and DNA Profiling Genome in a Bottle Consortium Workshop August 2014 Don A. Baldwin, Ph.D. CSO, Pathonomics LLC
    2. 2. ABRF is an international organization of over 700 scientists from shared research resource core facilities and biotechnology laboratories. Members represent over 250 core labs in academic and research institutions, government, and industry. “Yellow pages” and “MarketPlace” databases of members at www.ABRF.org Electronic discussion group facilitates sharing of technical advice and core facility networking. The Journal of Biomolecular Techniques covers genomics, proteomics, imaging, and other biotechnologies, and core facility operational management.
    3. 3. www.abrf.org Metagenomics (MGRG)
    4. 4. The ABRF Next Generation Sequencing (NGS) Study: • Produce reference data sets to establish baseline performance • Promote the use of standard samples • Provide public access to data for self-evaluation, performance monitoring and methods development Phase I: RNA-Seq and degraded RNA-Seq (2011-2013) Phase II: DNA-Seq and hard-to-sequence regions and samples (2014-2016) Phase III: Clinical genetics sequencing panels
    5. 5. Phase I Study Design
    6. 6. Major Conclusions Intraplatform concordance: Spearman rank R > 0.86 Interplatform concordance: R > 0.83 Q10 – Q60, most variation at read starts and ends Higher alignment rates with platform-specific algorithms vs. STAR Higher single-base mismatch and indel rates with platform- specific algorithms vs. STAR Wide range of efficiencies and costs for splice junction profiling Highly similar profiles from rRNA-depleted and polyA-enriched samples Effective analysis of degraded RNA after rRNA depletion
    7. 7. Funded by: Vendor donations of sample preparation and sequencing reagents Participating laboratories ABRF Nature Biotechnology, September 2014 6 figures, 2 tables 39 supplementary figures, 7 supplementary tables The ABRF NGS Study, Phase I 26 primary scientists 34 contributing scientists 21 research institutions 4.3 billion reads 447 billion nucleotides
    8. 8. The ABRF NGS Study, Phase II DNA sequencing topics were brainstormed and prioritized by the study consortium Samples were chosen based on the August 2013 Genome in a Bottle Workshop
    9. 9. Phase II DNA sequencing aims Reference data sets • Intra- and inter-lab replication to model the range of performance expected under normal service laboratory conditions Reference samples • Easily accessible for self-evaluation by comparison to the reference data • Standardized, stably reproduced, suitable for methods development Immediate utility • Performance metrics and data applicable to methods used now or in the near future by sequencing core facilities
    10. 10. Projects in no particular order, with project scope and sequencing coverage to be prioritized by interest and funding: Performance using different platforms and technical protocols • NIST GiaB designated human genomic DNA • Measure sequencing accuracy and coverage Performance using damaged DNA and chimeric cell populations • DNA from formalin-fixed, paraffin embedded cells • Measure sequencing accuracy, coverage, and limits of detection for somatic mutations Performance on small genomes over a range of GC content • NIST GiaB (with FDA) designated bacterial genomic DNA • Measure sequencing accuracy and coverage
    11. 11. Samples Sample ID DNA source Sequencing Project Per replicate Breadth Depth A Ashkenazim Jew PGP, maternal cell line 1 genome 35x B Ashkenazim paternal cell line 1 genome 35x C Ashkenazim child cell line from NIST stock 1 genome 35x Performance using different platforms and technical protocols
    12. 12. Performance using damaged DNA and chimeric cell populations Sample ID DNA source Sequencing Project Per replicate Breadth Depth M pool of FFPE DNA from mutant AcroMetrix lines #1, #2 and #3 plus Horizon Dx line #4: 1 and 4 40% each, 2 and 3 10% each by copy number Syn Accugenomics pool of synthetic templates for the mutations in lines #1-#4; tagged, stock = 40:40:10:10 C2 Ashkenazim child cell line from Coriell stock 2 exome 100x C2f Ashkenazim child cell line suspension, formalin-fixed, paraffin embedded 2 exome 100x Mf0 100% M DNA from FFPE, spike gDNA with Syn at molarity = single copy gene in total M DNA 2 exome 100x Mf1 25% C2f, 75% M (each target’s copy number = 15% or 3.75%); spike gDNA with Syn = M 2 exome 100x Mf2 50% C2f, 50% M (targets = 10% or 2.5%); spike gDNA with Syn = M 2 exome 100x Mf3 75% C2f, 25% M (targets = 5% or 1.25%); spike gDNA with Syn = M 2 exome 100x Mf4 90% C2f, 10% M (targets = 2% or 0.5%); spike gDNA with Syn = M 2 exome 100x Mf5 95% C2f, 5% M (targets = 1% or 0.25%); spike gDNA with Syn = M 2 exome 100x Mf6 99% C2f, 1% M (targets = 0.2% or 0.05%) Mf7 99.5% C2f, 0.5% M (targets = 0.1% or 0.025%) Mf8 99.9% C2f, 0.1% M (targets = 0.02% or 0.005%) Samples
    13. 13. Oncogenic mutations • BRAF V600E • KRAS G12C • EGFR c.2235_2249 del15 • EML4-ALK
    14. 14. Sample ID DNA source Sequencing Project Per replicate Breadth Depth Sta Staphylococcus aureus 3 genome 100x Sae Salmonella enterica 3 genome 100x Psa Pseudomonas aeruginosa 3 genome 100x Cls Clostridium sporogenes 3 genome 100x P pooled metagenomic sample with all four bacterial genomes 3 genome 100x Performance on small genomes over a range of GC content Samples
    15. 15. Species Genome (bp) Avg % GC Reference strain Distributor Staphylococcus aureus 2.8x106 33 NRS77 (NCTC 8325) NARSA #NRS77 Salmonella enterica subsp. enterica serovar Typhimurium 4.9x106 52 LT2 ATCC #700720 Pseudomonas aeruginosa 6.7x106 67 PA01 ATCC #47085 Clostridium sporogenes 4.1x106 28 Metchnikoff ATCC #15579 Small genomes project: sizes and GC content
    16. 16. Platforms and library methods Platform Project 1 Samples Project 2 Samples Project 3 Samples Illumina X10 A, B, C, C2 Illumina 1 T A, B, C, C2 Illumina NextSeq 500 A, B, C, C2 Sta, Sae, Psa, Cls, P Illumina HiSeq 2500 A, B, C, C2 C2, C2f, Mf0-Mf5 Illumina 2500 Rapid run C for long-read scaffold Illumina MiSeq C for long-read scaffold Sta, Sae, Psa, Cls, P Life Technologies Proton A, B, C C2, C2f, Mf0-Mf5 Sta, Sae, Psa, Cls, P Life Technologies PGM Sta, Sae, Psa, Cls, P Pacific Biosciences C for long-read scaffold Sta, Sae, Psa, Cls, P Qiagen GeneReader Sta, Sae, Psa, Cls, P Library Protocol Illumina Moleculo A, B, C Nextera on HiSeq A, B, C Sta, Sae, Psa, Cls NuGEN on HiSeq A, B, C Sta, Sae, Psa, Cls New England Biolabs on HiSeq A, B, C Sta, Sae, Psa, Cls Kapa on HiSeq A, B, C Sta, Sae, Psa, Cls Rubicon on HiSeq A, B, C Sta, Sae, Psa, Cls Bioo on HiSeq A, B, C Sta, Sae, Psa, Cls EXOME: Agilent Sure Select C2, C2f, Mf0-Mf5 EXOME: Roche Nimblegen SeqCap EZ C2, C2f, Mf0-Mf5 EXOME: Ampliseq Exome Panel for Proton C2, C2f, Mf0-Mf5
    17. 17. An ABRF – GiaB collaboration • Get vendor commitments for technical support and reagent donations • Extract high-quality genomic DNA from cultured cells for A, B, C, C2, Sta, Sae, Psa and Cls • Prepare equimolar blend of bacterial DNA for pool P • Procure somatic mutation cell lines in FFPE blocks • Extract genomic DNA from FFPE blocks of cell suspensions, prepare blends • Assemble platform groups with at least 3 labs per instrument or method • Each platform group will determine a consensus protocol for library preparation and sequencing • Distribute aliquots of DNA reference stocks to participating study labs • Construct and/or sequence libraries (intra-lab replicates encouraged) • Collect and annotate data in a central repository • Analyze sequencing performance planned started complete
    18. 18. Name email Contact regarding: Baldwin, Don donabaldwin65@yahoo.com study design Grills, George gsg34@cornell.edu vendor and partner relations Mason, Chris chm2042@med.cornell.edu data analysis Nicolet, Charlie cnicolet@usc.edu sequencing methods Tighe, Scott scott.tighe@uvm.edu logistics The ABRF NGS Study leadership group in alphabetical order, with level of participation and devotion to be prioritized by alcoholic intake:

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