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Similar to Building a platinum human genome assembly from single haplotype human genomes generated from long molecule sequencing
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Building a platinum human genome assembly from single haplotype human genomes generated from long molecule sequencing
- 1. Building a platinum human genome assembly from single haplotype human genomes generated from long molecule sequencing Karyn Meltz Steinberg ASHG 2015 @KMS_Meltzy
- 2. 0 100000 200000 300000 400000 CHM1_1.1 HuRef ALLPATHS YH_2.0 Contig Number Contig N50 Figure 1 Last year… Steinberg et al, 2014
- 3. This year… 0 5000000 10000000 15000000 20000000 25000000 30000000 CHM13 Draft CHM1 PB_2 CHM1 PB_1 CHM1_1.1 HuRef ALLPATHS YH_2.0 Contig Number Contig N50
- 4. This year… Log scale 1 10 100 1000 10000 100000 1000000 10000000 100000000 CHM13 Draft CHM1 PB_2 CHM1 PB_1 CHM1_1.1 HuRef ALLPATHS YH_2.0 Contig Number Contig N50
- 5. We combine PacBio with other technologies to construct the assembly
- 6. How do we define platinum and gold standards? GRCh38 Platinum (CHM1) Gold (NA19240) % Reference genome covered 100 98.40 90.80 % Assigned chromosomes 99.60 98.40 90.80 % gene models covered (>95% id, >90% length) 99.96 98.78 94.26 Contig N50 67.8 Mb 26.9 Mb 6.0 Mb Number of gaps 875 3,640 3,568 Total Assembled size 3.067 Gb 2.996 Gb 2.745 Gb % haplotype blocks (>1kb) resolved NA >95 >80 http://genome.wustl.edu/projects/detail/reference-genomes-improvement/
- 7. CHM13 Draft Assembly (GCA_000983455.1) • 60X PacBio (P5 and P6 chemistry) • Average read length ~11kb • Daligner/Falcon v 0.2 Total sequence length 2,851,367,788 Number of contigs 2,873 Contig N50 12,981,785 Contig L50 68
- 8. Gene Model (RefSeq) Analysis GRCh38 CHM1_ 1.1 CHM1_PB1 CHM1_PB2 CHM13 Number of sequences not aligning 21 88 67 67 125 Split Transcripts 8 35 1,245 1,131 285 CDS coverage <95% 17 266 1,339 1,212 265 Total Sequences Retrieved from Entrez 49,680
- 9. Short read sequence analysis • 100X Illumina sequence • Align with BWA-MEM to ordered and oriented assembly • Variant calling via SpeedSeq (Chiang et al, 2015) • SNVs, indels: FreeBayes • SVs: LUMPY, SVTyper • CNV: CNVnator
- 10. CHM13 Illumina data aligned to CHM13 assembly 202,016 SNVs/indels on unplaced scaffolds SV_TYPES >10kb 5-10kb 1-5kb <1kb DELETIONS 174 131 430 2582 INVERSIONS 5 0 2 7 DUPLICATIONS 151 112 309 113 TOTAL 330 243 741 2702
- 11. BioNano SV calls can be used to identify misassembly Collapse Expansion inAssembly Gap in SequencePacBio Assembly BioNano Map SV_TYPES DELETIONS 41 INVERSIONS 10 INSERTIONS 15 TOTAL 66 BioNano alignment to CHM13
- 12. BioNano reveals collapse in PacBio assembly PacBio Assembly BioNano Map
- 13. Illumina data aligned to PacBio assembly also shows collapse
- 14. BioNano reveals collapse in PacBio assembly due to highly homologous segmental duplications SD = 96% CHR1 46746040 46857004 40 W LBHZ01000938.1 110965 CHR1 46857005 47034202 41 N 177198 gap CHR1 47034203 52157695 42 W LBHZ01000245.1 5123493 PacBio Assembly BioNano Map
- 15. This region is rich in medically relevant genes chr1 (p33) p31.1 1q12 q41 43 44 CYP4Z2P CYP4A11 CYP4X1 CYP4Z1 CYP4A22 SegDups Genes CHM13 PacBio LBHZ010000938.1 LBHZ010000938.1 LBHZ010000245.1
- 16. CHM13 Hybrid Scaffold Hybrid Scaffold PacBio Contigs BioNano Contigs
- 17. CHM13 Hybrid Scaffolds BioNano Map PacBio Assmbly Hybrid Scaffold # of Contigs 3593 1590 * 254 Min Contig Length 0.08 Mb 0 0.27 Mb Median Contig Length 0.61 Mb 0.06 Mb 4.35 Mb Mean Contig Length 0.78 Mb 1.78 Mb 9.68 Mb Contig N50 1.02 Mb 13.46 Mb 20.79 Mb Max Contig Length 5.27 Mb 63.15 Mb 82.83 Mb Total Contig Length 2.812 Gb 2.824 Gb 2.458 Gb *Number of contigs used in hybrid scaffolding
- 18. Combining CHM1 and CHM13 reference mapping CHM1 CHM13 Pipeline analysis Variant Evaluation 97 13
- 19. Acknowledgements The McDonnell Genome Institute at Washington University in St. Louis Rick Wilson Bob Fulton Wes Warren Tina Graves-Lindsay Vince Magrini Sean McGrath Derek Albracht Milinn Kremitzki Susan Rock Debbie Scheer Aye Wollam The Finishing and Bioinformatics Teams at The Genome Institute University of Washington Evan Eichler John Huddleston Archana Raja NCBI Valerie Schneider University of Pittsburgh School of Medicine (CHM13 cell line) Urvashi Surti Personalis Deanna Church BioNano Genomics Palak Sheth Pacific Biosciences Jason Chin Nick Sisneros