F Giordano ScanPAV Analysis Pipeline
•
0 likes•53 views
Description of the ScanPAV pipeline https://github.com/wtsi-hpag/scanPAV
Recommended
Recommended
More Related Content
What's hot
What's hot (20)
Similar to F Giordano ScanPAV Analysis Pipeline
Similar to F Giordano ScanPAV Analysis Pipeline (20)
More from Francesca Giordano
More from Francesca Giordano (12)
Recently uploaded
Recently uploaded (20)
F Giordano ScanPAV Analysis Pipeline
- 1. 1 ScanPAV: Presence-Absence Variation in Genome Pairs Francesca Giordano IGM, November 7th, 2017
- 2. Estimation of Genetic Divergence ‣ As important as SNPs/indels, CNVs to estimate genetic variations ‣ Provides insights into population or cancer evolution ‣ Helps identifying foreign DNA contamination ‣ Helps identifying lateral transfer of sequences from viruses or bacteria 2 Presence-Absence Variation (PAV) Assembly Strategy and Technology Assessment ‣ Determines the completeness of an assembly ‣ Helps assessing an assembly strategy and pipeline ‣ Helps identifying strengths and weaknesses in new sequencing technologies
- 3. 3 Presence Assembly ScanPAV: pipeline 1 Kb Presence Assembly Shred into 1Kb chunks
- 4. 3 Presence Assembly AbsenceAssembly Scaffold 1 Scaffold 2 Scaffold 3 ScanPAV: pipeline 1 Kb Presence Assembly Shred into 1Kb chunks [SMALT Aligner] Map Against Absence Assembly
- 5. 3 Presence Assembly AbsenceAssembly Scaffold 1 Scaffold 2 Scaffold 3 ScanPAV: pipeline 1 Kb Presence Assembly Shred into 1Kb chunks [SMALT Aligner] Map Against Absence Assembly Filter noisy mapping
- 6. 3 Presence Assembly AbsenceAssembly Scaffold 1 Scaffold 2 Scaffold 3 ScanPAV: pipeline 1 Kb Presence Assembly Shred into 1Kb chunks [SMALT Aligner] Map Against Absence Assembly Output Not Mapped Areas Filter noisy mapping
- 7. Human Assemblies ‣ HuRef (2007), 7.5x whole-genome shotgun Sanger 800 bp PE reads, Celera [Levy S, et al., 2007, PLOS Biology 5(10), e254] ‣ Hs2-HiC (2017) 60x Illumina 250 bp PE reads, DISCOVAR denovo, Hi-C data for scaffolding [Dudchenko O. et al., 2017, Science, doi: 10.1126/science.aal3327] ‣ Illumina (2016) 39x short-insert and 24x long-insert Illumina 101 bp PE reads, spaDES, 10X genomics and Bionano data for scaffolding [Mostovoy Y. et al., 2016, Nature Methods, 13, 587] ‣ AK1 (2016) 101x PacBio long reads (mean 10Kb), Falcon, polished with Quiver and Pilon (Illumina), Bionano maps for scaffolding [Seo J.S. et al., 2016, Nature, 538, 243] ‣ ONT_30x (2017) 30x Oxford Nanopore long reads (mean 11Kb), Canu, polished with Pilon (Illumina). No scaffolds [Jain, M. et al., 2017, bioRxiv, doi:10.1101/128835] ‣ ONT_35x (2017) Same as ONT_30x plus 5x extra-long Oxford Nanopore reads (n50 65Kb), Canu, polished with Pilon (Illumina). No scaffolds [Jain, M. et al., 2017, bioRxiv, doi:10.1101/128835] 4
- 8. Human Assemblies 5 Assembly #Bases (Gb) #Contigs/ Scaffolds Longest (Mb) Contig-n50 (Mb) Scaffold-n50 (Mb) GRCh38 3.1 24 249 59 156 HuRef 2.8 3,134 235 0.11 144 AK1 2.9 2,832 114 18 45 Hs2-HiC 2.8 44,065 225 0.10 141 Illumina 2.9 170 100 0.01 33 ONT_30x 2.8 2,886 28 4 — ONT_35x 2.9 2,337 50 8 —
- 9. Human Assemblies 5 Assembly #Bases (Gb) #Contigs/ Scaffolds Longest (Mb) Contig-n50 (Mb) Scaffold-n50 (Mb) GRCh38 3.1 24 249 59 156 HuRef 2.8 3,134 235 0.11 144 AK1 2.9 2,832 114 18 45 Hs2-HiC 2.8 44,065 225 0.10 141 Illumina 2.9 170 100 0.01 33 ONT_30x 2.8 2,886 28 4 — ONT_35x 2.9 2,337 50 8 — 800 bp 250 bp 101 bp
- 10. ScanPAV analysis 6 PAVs (Mb) Present in: PAVs (Mb) Absent in: GRCh38 HuRef AK1 Hs2-HiC Illumina ONT_30x ONT_35x GRCh38 0 33 20 35 263 65 55 HuRef 18 0 26 33 197 67 58 AK1 14 33 0 32 225 62 54 Hs2-HiC 23 34 27 0 200 59 49 Illumina 91 109 98 95 0 124 117 ONT_30x 21 40 27 29 183 0 35 ONT_35x 47 64 47 53 268 61 0
- 11. ONT_35x: Homomer Counts 7 TTTTTT AAAAAA CCCCCC GGGGGG Draft Presence−PAVs Absence−PAVs 0.00.51.01.52.02.53.0 6−mers 6−homopolymerfrequency Homopolymer frequency / Draft frequency
- 12. Mis-joints analysis 8 Map chr-level scaffolds Against Reference
- 13. Mis-joints analysis 8 Map chr-level scaffolds Against Reference If no chr-level scaffolds: assign ctgs to chrs
- 14. Mis-joints analysis 8 Map chr-level scaffolds Against Reference If scaffold map to more than 1 chrs If no chr-level scaffolds: assign ctgs to chrs
- 15. Mis-joints analysis 8 Map chr-level scaffolds Against Reference If scaffold map to more than 1 chrs Misjoint If no chr-level scaffolds: assign ctgs to chrs
- 16. Mis-joints analysis 8 Map chr-level scaffolds Against Reference If scaffold map to more than 1 chrs Misjoint If no chr-level scaffolds: assign ctgs to chrs AK1 GRCh38 Chr16Chr2 SY-16SY-2
- 17. Mis-joints analysis 9 Scaffold Assigned Chr Mis-Joints (>300 Kb) Length (bp) Chr Avg id (%) HuRef — Hs2-HiC Hs2-HiC_hic_scaffold_9 19 300 K 22 99.0 Hs2-HiC_hic_scaffold_10 21 1.3 M 22 99.3 AK1 synteny_group_16 16 16 M 2 99.7 ONT_35x synteny_group_1 1 3.9 M 19 99.8 synteny_group_1 1 565 K 3 99.0 synteny_group_2 2 1.8 M 1 99.1 synteny_group_4 4 316 K 20 99.1 synteny_group_4 4 543 K 5 99.2 synteny_group_5 5 2.2 M 18 99.1 synteny_group_6 6 3.3 M 7 99.1 synteny_group_8 8 3.7 M 16 98.9 synteny_group_10 10 5.8 M 1 99.0 synteny_group_10 10 2.0 M 11 98.9 synteny_group_12 12 13.9 M 10 99.1 synteny_group_13 13 4.2 M 10 99.1 synteny_group_15 15 2.0 M 3 98.8 synteny_group_16 16 1.4 M 1 98.8 synteny_group_22 22 342 K 19 98.6
- 18. 4 Fig. S2. Misplaced block visualisation for chromosome assigned scaffolds in Hs2-HiC (left), AK1 (center) and ONT_35x (right). Table S3. Misjoint location in the original scaffolds for assemblies AK1 and ONT_35x. Assembly Mis- Joint Original Scaffold 1st Chromosome Length mapped to 1st Chromosome Break Position 2nd Chromosom Giordano et a sation for chromosome assigned scaffolds in Hs2-HiC (left), AK1 (center) and ONT_35x (right). on in the original scaffolds for assemblies AK1 and ONT_35x. Original Scaffold 1st Length mapped to st Break 2nd Length mapped to nd Mis-joints analysis 10 G Hs2-HiC AK1 ONT_35x
- 19. Mis-joints analysis 11 Scaffold First Chr Breaking Area Second Chr Chr Length (bp) Chr Length (bp) AK1 KV784727.1 16 16.9 M 16,390,000 – 16,390,001 2 16.3 M ONT_35x tig00001490_pilon_pilon 1 5.5 M 3,999,036 – 4,000,001 19 3.9 M tig01414718_pilon_pilon 1 23.6 M 23,710,000 – 23,713,101 3 561 K tig00000928_pilon_pilon 1 2.0 M 23,710,000 – 23,713,101 2 3.0 M tig00000326_pilon_pilon 4 10.2 M 330,000 – 365,771 20 322 K tig01414909_pilon_pilon 4 9.5 M 557,558 – 560,001 5 535 K tig01415181_pilon_pilon 5 9.2 M 9,740,000 – 9,741,384 18 2.2 M tig00000726_pilon_pilon 6 6.9 M 3,399,916 – 3,400,054 7 3.4 M tig00001250_pilon_pilon 8 4.7 M 4,820,000 – 4,820,001 16 3.7 M tig01414799_pilon_pilon 10 14.4 M 14,700,000 – 14,770,556 1 5.8 M tig01415009_pilon_pilon 10 9.1 M 2,037,882 – 2,040,001 11 2.0 M tig01414760_pilon_pilon 12 18.0 M 18,455,260 – 18,460,001 10 13.9 M tig01414699_pilon_pilon 13 45.2 M 46,129,991 – 46,131,368 10 4.2 M tig00002429_pilon_pilon 15 7.3 M 7,426,592 – 7,430,001 3 2.0 M tig00001215_pilon_pilon 16 3.9 M 1,719,551 – 2,010,001 1 1.6 M tig00000735_pilon_pilon 22 11.0 M 358,160 – 360,002 19 352 K
- 20. Tasmanian Devil’s Transmissible Cancer 12 First Observed in NE Tasmania in 1996 Tumour- Free Area Devil Facial Tumour (DFT): ‣ Large tumour growths on face and neck ‣ Transmissible by bite ‣ Affect mostly adult individuals ‣ Death occurs in 4-6 months ‣ Two genetically different Cancers with similar phenotypes: DFT1 and DFT2
- 21. Tasmanian Devil’s Transmissible Cancer 13 A comparative characterisation analysis for DFT1 and DFT2 has been submitted to Science* recently [Stammnitz M.R. et al., The origins and vulnerabilities of two transmissible cancers in Tasmanian devils] PAV estimation is one of the numerous analyses presented, both to assess assembly completeness and to look for exposure to exogenous agents that might have contributed to the cancer development. The scanPAV analysis involved: ‣ 2 Existing References: Ref-v7.1 and PSU ‣ 6 New Assemblies: 2 from healthy cells: 202H, 203H 4 from tumour cells: 202T, 203T, 86T, 88T [Stammnitz M.R. et al., The origins and vulnerabilities of two transmissible cancers in Tasmanian devils, in review @ Science]
- 22. Devil’s PAVs 14 The extracted PAVs sequences were screened against the NCBI database: - no foreign DNA found in the references - in 202T: found 590 Kb sequence belonging to Mycoplasma arginini genome - All 6 new assemblies PAVs contained 1.9 Mb of the Streptococcus pneumoniae genome Both well known laboratory cell culture contaminants Remaining PAVs are believed to be due to assembly errors and ancestral variation No exogenous contribution to the emergence of DFTs found [Stammnitz M.R. et al., The origins and vulnerabilities of two transmissible cancers in Tasmanian devils, in review @ Science]
- 23. Summary ‣ Assessing the completeness and quality of a newly generated genome assembly is a complex task and requires evaluating multiple metrics. In presence of multiple assemblies or of a reference assembly: ‣ the extraction of PAV sequences can help assessing strengths and weaknesses of a novel assembly strategy or technology, as well as identifying structure variation and foreign DNA exposure ‣ indication of possible mis-joints can be inferred by mapping the assembly to the reference and identify scaffolds that map to multiple chromosomes ‣ scanPAV pipeline for pair-wise assembly extraction of PAVs is available @ https://sourceforge.net/projects/phusion2/files/scanPAV ‣ In progress: including BWA as an alternative aligner 15
- 24. 16 Acknowledgments Paul A. Kitts, National Center for Biotechnology Information, Bethesda, MD Elizabeth P. Murchison, University of Cambridge, Cambridge, UK Zemin Ning, Wellcome Trust Sanger Institute, Hinxton, UK Maximilian R. Stammnitz, University of Cambridge, Cambridge, UK Thank you!
- 25. Backup Slides 17
- 26. Devil Assemblies 18 Assembly #Bases (Gb) Scaffolds (103) Longest (Mb) Contig-n50 (Kb) Scaffold-n50 (Mb) Ref-v7.1 3.2 36 5.3 20 1.8 PSU 3.2 149 2.9 11 0.2 202T2 3.0 62 50.7 55 9.5 202H1 3.0 68 22.0 51 4.2 203T3 3.0 63 50.7 53 9.6 203H 3.0 64 31.1 48 4.2 86T 3.0 70 24.5 66 4.4 88H 3.0 61 19.7 60 4.0 [Stammnitz M.R. et al., The origins and vulnerabilities of two transmissible cancers in Tasmanian devils, in review @ Science]
- 27. 19 PAVs present in: PAVs (Mb) Absent in: Ref-v7.1 PSU 202T2 202H1 203T3 203H 86T 88T Ref-v7.1 0 156 62 67 61 64 55 58 PSU 125 0 69 76 69 71 63 66 202T2 110 149 0 36 22 28 26 25 202H1 107 147 28 0 29 27 28 27 203T3 107 146 20 34 0 27 24 24 203H 109 148 25 32 27 0 26 25 86T 106 146 28 38 29 31 0 22 88H 106 146 25 35 26 27 19 0 Devil’s PAVs
- 28. Devil’s Filtered PAVs 20 PAVs present in: PAVs (Mb) Absent in: Ref-v7.1 All Filtered PSU 125 5 202T2 110 14 202H1 107 11 203T3 107 12 202H 109 12 86T 106 12 88H 106 13 PAV filter: filter out sequences missing from Absent Assembly but present in reads Absent Assembly + PAVs Absent Assembly Reads Mapped Filtered PAVs Filter out PAVs with 10x depth Most of the missing sequences are present in the Genome (reads), but Absent in the assembly [Stammnitz M.R. et al., The origins and vulnerabilities of two transmissible cancers in Tasmanian devils, in review @ Science]