Optimizing Blue Pippin Size- Selection for Increased SubRead Lengths on the RSII Applications in Clinical Genomics
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1 Optimizing Blue Pippin Size- Selection for Increased SubRead Lengths on the RSII Applications in Clinical Genomics Robert P. Sebra, Ph.D. Icahn School of Medicine at Mount Sinai Icahn Institute for Genomics & Multi-scale
2 An Integrated Omics Approach
3 Requires a Multi-faceted Sequencing Technology Sandbox Multi-Platform DNA Sequencing First Generation 1 x Applied Biosystems 3730xl Second Generation 5 x Illumina HiSeq x MiSeq 1 x Ion Proton 2 x Ion PGM Third Generation 2 x PacBio RS II Microarray Genotyping and Expression 1 x Illumina Bead Array platform Liquid Handling Automation 1 x Agilent Bravo 2 x Tecan EBO 1 x Beckman FX Robot Local IT Infrastructure 100 Tb mirrored primary storage 1,500 Tb secondary storage
4 The SMRT Sequencing Program at Mount Sinai GCF Human Sequencing -Complex genetic loci (TR, CNV, rearrangements, etc.) -Exploring intronic space -Clinical validation -Allelic phasing -High resolution genotyping -Targeted sequencing Infectious Disease -Hospital surveillance -Rapid microbial finishing -Phasing plasmids -Building phylogenies -Metagenomics -Understanding co-infection RSII Upgrades & Sage Blue Pippin SS 12-month Highlights Basic Research -Full length transcriptome -Basic genomics research -Novel Bifx pipelines -Reducing DNA input -Methods development for targeted/capture capability Epigenetics -mtdna disease -Discovering novel motifs -Growth phase comparisons -Virulence factors -Oxidative or photochemical damage associated w/ cancer ~1800 SMRTcells sequenced in the past 14 months -Throughput has increased by ~4X, but as much as 10X in some cases -subrl has increased by ~2X, but up to 5X
5 Size Selection using the Blue Pippin Technology I Input 10kb shear Input 20kb shear >5kb >10kb [DNA] Sheared DNA , 8614 Pippin High-Pass Selected Size (kb) Shear to 10kb avg. Shear to 20kb avg. Size select >5kb Size select >10kb PFGE view 5
6 Size Selection using the Blue Pippin Technology II 4 samples per run. Typical runtimes for DNA >10kb: 2-8 hours.
7 Applications Requiring Blue Pippin Size Selection at Sinai Large Insert Size Selection (0.75% Cassette) Short Insert Size Selection (2.0% Cassette) -Removal of fragments <10kb -Purification of specific DNA (mtdna) -Selection of large plasmids Maximize library size for longest N50 subreads Removal of <10kb library -Amplicon or digested DNA selection (by size) -Purification of short libraries -Plasmid selection < 10kb Sample purity Selected bp
8 Pipelines Standard Pipeline Obtain tissue, cells, or swab for generating gdna OR Culture colony to generate the appropriate mass Isolate 6ug gdna (various methods) Plasmid (<10kb) Pipeline Use 5ug gdna or Shear to ~20kb 20kb+ SMRTbell Library Construction 10ng to WGA if control needed Shear 1ug to 2kb-6kb Tet Convert, if desired Blue Pippin Size Select at >7 or 10kb Option: Mix w/ Plasmid Library Sequencing using 50 pm P4-C2, 120 MB collection HGAP De Novo Assembly & Variant Analysis 2kb SMRTbell Library Construction Blue Pippin Size Select if needed Sequencing using 20 pm P4-C2, 120 MB collection Plasmids Sequenced and/or Base Modification Profile
9 Blue Pippin Size Selection Performance: Yield & Capacity 67 BPSS libraries 100 SMRTcells 40 SMRTcells 10 SMRTcells 2 SMRTcells -Only 1 SS library has insufficient yield for 2 chips -Average % yield (by mass) = 24% -Yield is dependent on input DNA size distribution but parameters can be adjusted accordingly (7kb vs 10kb) Tips for Maximizing Yield: 1. Use DNA isolation techniques with gentle steps (Qiagen Tip works well) 2. Conduct AMPure prior to shearing to remove small fragments for true assessment of input mass. 3. Remove any bubbles in cassette by tapping, etc. 4. Wash all loading & elution wells with E-buffer prior to placing sample 5. If bubble is in well, don t use that well. 6. After sample collection, rinse all elution wells w/ 40uL of E-buffer to collect DNA
10 Blue Pippin Size Selection Impact on SubRL Distributions % Sequenced Bases w/o BPSS: Impact of BPSS on N50 Length (MRSA) 95 th % ~ 12,400bp N50 ~ 5000bp Best Read ~21,000bp ~240Mb mapped 1 SMRTcell ~5kb ~12.5kb w/ BPSS: subrl Threshold 95 th % ~ 19,700bp N50 ~ 12,500bp Best Read ~34,500bp ~325Mb mapped / cell 2 SMRTcells
11 Rapid & Cost Effective Infectious Disease Surveillance Example: MRSA -Main culprit in healthcare-associated infections -Regulatory agencies now require mandatory reporting, reimbursements held -At Sinai, each positive blood stream culture requires a consult due to: High mortality rates High treatment failure rates/relapses Drug resistance -Accounts for nearly half of all inpatient Infectious Diseases consultations Patient Cultures Drawn PacBio RSII sequencing & HGAP assembly + Cultures inoculated on agar plates in MicroLab Colonies identified by routine MicroLab techniques Pipeline: From Bedside to Sequencing gdna QC & sample preparation (previously shown) ~ 48 hours & <$300 DNA isolated using Qiagen DNATip (or similar kit) Colonies streaked for single colony & single colony grown Cells harvested, spun, and lysed for DNA isolation
12 Examples of Microbial Assemblies after BPSS / RSII # of SMTcells # of Reads Mean subrl 95 th % RL Coverage Assembled Bases N50 of Contigs Largest Contig MRSA ,000 2,986 12, X 2.8 Mb 47, , MRSA ,010 3,787 13, X 2.98 Mb 300, , MRSA ,032 7,088 19, X 2.90 Mb 1.96 Mb 1.96 Mb 11 MRSA ,106 5,787 15, X 2.92 Mb 2.92 Mb 2.92 Mb 2 MRSA ,253 7,485 20, X 2.94 Mb 2.94 Mb 2.94 Mb 1 Bacteria ,239 5,507 14, X 4.75 Mb 4.73 Mb 4.73 Mb 3 Bacteria ,140 5,424 14, X 4.53 Mb 4.53 Mb 4.53 Mb 1 Bacteria ,322 4,868 13, X 4.83 Mb 3.46 Mb 3.46 Mb 10 Bacteria ,612 5,578 13, X 4.78 Mb 799, Mb 18 Bacteria ,349 5,758 13, X 4.71 Mb 4.69 Mb 4.69 Mb 2 -Complete microbial genomes in as little as 1-4 SMRT Cells -Some contigs may be plasmids -MRSA 1 is reduced from 87 to 11 contigs w/ BPSS (Others to a single contig) -DNA quality (from bead beating conditions and/or prep) is important -All assemblies stats from HGAP 2.0.x (no partial alignments) # Contigs
13 10X Mapped Coverage of Human Genome NA12878 Single and Paired End Illumina Reads PacBio Reads ~100s of M Number of reads Number of Reads ~12M Mapped coverage ~30X Mapped coverage 10X+ 454 Reads Number of reads ~100M Mapped coverage ~15X Mean sub-read length 2,766 Mean unrolled read length 4, th Percentile 11,630 Accuracy (error-corrected reads) >99%
14 Hard to Sequence Human Genes w/ Disease Association Genes involving TNR expansions Panel of genes we re interested in for inherited disease screening
15 CACNA1A gene TNRs Associated w/ Spinocerebellar Ataxia 6 Calcium channel, voltagedependent, P/Q type, alpha 1A subunit gene Arrow denotes read spanning exons to link separate TNRs chr19: chr19: ~10-15X reads span both TNRs Variants sssociated with familial hemiplegic migraine and episodic ataxia CAG ACC
16 Long Reads Suggest Under Representation of TR Spans in Reference Reference suggests bias towards repeat length compression
17 Impact of BPSS & Longer ReadLength Chemistry on NA12878 CLR Means w/ & w/o Blue Pippin Size Select Mean sub-rl w/o Pippin SS Mean sub-rl w/ Pippin SS 2,766 bp 4,491 bp 95 th Percentile w/o Pippin SS 11,630 bp 95 th Percentile w/ Pippin SS 13,266 bp Standard 10-20kb library Size Selected 10-20kb library ~82Mb > 5kbp Fragments < 7kb eliminated ~22Mb > 10kbp Size Select Protocol was set to select 7kb-50kb -Blue Pippin electrophoretic size selection improves subrl by 62% by removing DNA fragments < 7kb to avoid small molecule loading bias.
18 Impact of BPSS & Longer ReadLength Chemistry on NA February Min. : 40 1st Qu.: 974 Mean : rd Qu.: 2739 Max. :21458 SubRL Statistics August Min. : 40 1st Qu.: 1324 Mean : rd Qu.: 5855 Max. :30474 Present Min. : 40 1st Qu.: 1907 Mean : rd Qu.: 8211 Max. : SubRL (bp) Currently, we generate ~1X NA12878 coverage in 8-10 SMRTcells for ~$1000
19 Thanks! Sinai Team Deena Altman Ali Bashir Imane Bourgui Gintaras Deikus Andrew Kasarskis Alona Keren-Paz Milind Mahajan Eric Schadt Anne Schaefer Harm van Bakel Ajay Ummat Cornell Roger Altman Russell Durrett Chris Mason CSHL Eric Antoniou Richard McCombie Patricia Mocombe NYGC/Rockefeller Bob Darnell NYU Bo Shopsin Pacbio Jason Chin Ellen Paxinos PacBio Field Staff Sage Science
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