Using Long Reads, Optical Maps and Long-Range Scaffolding to improve the Diaphorina citri Genome: An update
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Improving Diaci1.1 genome with Pacbio, Dovetail, 10X and BioNano technologies
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Using Long Reads, Optical Maps and Long-Range Scaffolding to improve the Diaphorina citri Genome: An update
- 1. www.citrusgreening.org Using Long Reads, Optical Maps and Long- Range Scaffolding to improve the Diaphorina citri Genome: An update Surya Saha1, Susan J. Brown2 and Lukas Mueller1 1Boyce Thompson Institute; 2Kansas State Univeristy ss2489@cornell.edu @SahaSurya PAG XXV Arthropod Genomics Workshop
- 2. www.citrusgreening.org Acknowledgements Mueller Lab Mirella Flores Prashant Hosmani Kansas State University Sue Brown USDA/ARS Wayne Hunter
- 3. www.citrusgreening.org Citrus Greening: Huanglongbing • Most significant disease of citrus worldwide • More than $4.5 billion in lost citrus production and more than 8,200 lost jobs (2006/07 to 2010/11) • Associated with gram negative bacterium Candidatus Liberibacter asiaticus (CLas) • Spread by insect vector, Diaphorina citri (Asian citrus psyllid, ACP) Annie Kruse
- 4. www.citrusgreening.org Asian citrus psyllid (ACP) Diaphorina citri CLas Candidatus Liberibacter asiaticus Citrus spp. The Biological Players HostVector Pathogen
- 5. www.citrusgreening.org Current Assembly: Diaci v1.1 Data Source Library Count SRA Illumina Paired-end 99.7 mill SRX057205 Illumina 2kb Mate-pair 35.1 mill SRX057204 Illumina 5kb Mate-pair 30 mill SRX058250 Illumina 10kb Mate-pair 30 mill SRX216330 Pacbio 2.7 mill SRX218985 https://citrusgreening.org/organism/Diaphorina_citri/genome Input: Mixed sample of sexually reproducing heterogeneous individuals
- 6. www.citrusgreening.org Current Assembly: Diaci v1.1 Genome Scaffold N50:109,898 bp Contig N50: 34,407bp Highly fragmented Misassemblies Whitefly scaffold N50: 3.23 Mbp!! Genome Diaci1.1 Contigs 161,988 Total Length 485,705,082 or 485 Mb Longest 1,098,238 Shortest 201 Ns 19,337,167
- 7. www.citrusgreening.org 530 manually curated genes MCOT transcriptome to identify genes Many examples of misassemblies!! Preprint: http://biorxiv.org/content/early/2017/01/09/099168
- 8. www.citrusgreening.org Pacbio Sequencing 41 SMRT cells 70X coverage Counts Number of sequences 4,998,464 Total bases 36.1 Gb
- 9. DBG2OLC Hybrid Assemblyhttps://github.com/yechengxi/DBG2OLC NCBI Diaci1.1 and Pacbio assembly Very fast Adaptive Threshold Kmer Cov Threshold Contigs Total Length Longest Shortest Ns N50 (bp) 0.01 10 4,101 209,890,188 355,110 2,828 0 65,023 0.01 6 4,026 211,371,846 411,664 2,249 18 68,453 0.02 8 3,997 206,583,925 383,133 3,744 19 66,259
- 10. Miniasm Assembly (Raw Reads) https://github.com/lh3/miniasm/blob/master/README.md No error correction Very fast Contig N50: 83,490bp (was 34,407bp) Counts Number of contigs 8,060 Total bases 458,143,096 or 458 Mb Longest 1,188,453 bp Shortest 5, 633 bp Average length 56,841.6 bp
- 11. CANU Assembly http://canu.readthedocs.io/en/stable/ Error correction of 40% longest reads 26.5X coverage after correction Error rate 0.013 Error rate 0.015 Number of contigs 7,832 8,030 Total bases 462,838,769 or 462 Mb 493,169,880 or 493.1 Mb Longest 1,677,652 bp 1,757,402 bp Shortest 4,425 bp 5,079 bp Average length 59,095.9 bp 61,415.9 bp Contig N50 85,832 bp 92,630 bp
- 12. PBJelly Scaffolding of CANU Err 0.013 Assembly Error rate 0.013 Error rate 0.015 Scaffolded 0.03 Number of contigs 7,832 8,030 8,352 Total bases 462,838,769 or 462 Mb 493,169,880 or 493.1 Mb 591,730,999 bp or 591.7 Mb Longest 1,677,652 bp 1,757,402 bp 2,096,698 bp Shortest 4,425 bp 5,079 bp 1,547 bp Average length 59,095.9 bp 61,415.9 bp 70,849.0 bp Contig N50 85,832 bp 92,630 bp 115,896 bp 5,290 gap extensions; 535 gaps filled; Number of Ns: 0 bp
- 13. www.citrusgreening.org Benchmarking Complete Fragmented Missing Diaci 1.1 90% 6% 4% Diaci 1.9 92% 1% 7% White fly 98.2% 0.5% 1.3% PE RNAseq 622 Mill reads Overall alignment rate Concordant alignment rate Diaci 1.1 82% 0.62% Diaci 1.9 88% 79% Benchmarking sets of Universal Single-Copy Orthologs based on a set of 1,066 single-copy orthologs from 133 arthropods species
- 14. Diaci v1.9 Interim Assembly • Blast • FTP bulk download
- 15. www.citrusgreening.org FALCON-Unzip Assembler Hierarchical Genome Assembly Process (HGAP) Outputs • Primary contigs • Alternate contigs Haplotype resolution
- 16. www.citrusgreening.org Dovetail Scaffolding High molecular weight (50kb+) DNA 500ng input DNA from single male psyllid Illumina paired-end sequencing Chicago library preparation
- 17. www.citrusgreening.org Haplotyping Contigs with 10X Long read information from short reads using 14bp bar codes Very low input DNA (0.625 ng for ACP) 1ng of DNA is split across 100,000 Gel Coated Beads (GEMs) Chromium instrument http://www.10xgenomics.com/products/
- 19. www.citrusgreening.org Example: Human MHC map • Sample prep requires very high molecular weight DNA • Nicks at 10 sites / 100kb • Individual molecules are assembles into optical maps (Cmaps) • Optical maps and sequences are merged in a hybrid assembly http://www.bionanogenomics.com/technology/why-genome-mapping/ ACP molecule N50: 240 kb 1 Mb DNA fragments are ideal Optimizing enzymes for ACP
- 20. www.citrusgreening.org Twitter: 280 followers Facebook: 71 followers CitrusGreening.org Host, Vector and Pathogen(s) Blast Databases BioCycs JBrowse FTP site News Publications Social Media
- 21. www.citrusgreening.org P0368: Systems Biology Resources for the Citrusgreening Disease Complex Mirella Flores
- 22. www.citrusgreening.org Official Gene Set v1.0 530 manually curated genes 30,562 MCOT gene models Pathways: Developmental Physiological RNAi regulatory Immunity-related Preprint: http://biorxiv.org/content/early/2017/01/09/099168