Sequencing

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11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting TOWARDS BUILDING COMPLETE GENOME ASSEMBLIES USING BIONANO NEXT-GENERATION MAPPING TECHNOLOGY Thursday, 2nd June 17:25 La Fonda Ballroom Tech Talk (TT‐2.08) Andy Wing Chun Pang 1 , Thomas Anantharaman 1 , Xiang Zhou 1 , Jian Wang 1 , Joyce Lee 1 , Evan Eichler 2 , Tina Graves Lindsay 3 , Alex Hastie 1 , Han Cao 1 1 BioNano Genomics, 2 University of Washington, 3 Washington University High‐quality assemblies are important when trying to understand the biology of genomes. Current short‐read assemblers are memory intensive and have difficulties in constructing contiguous assemblies; collecting deep coverage data by long‐read technologies can be time‐consuming and expensive. BioNano’s Next‐Generation Mapping (NGM) data complements short‐read data by flagging and correcting inaccuracies and increasing contiguity, thereby reducing the need to collect high‐coverage long‐read data. BioNano Genomics Irys® System utilizes high‐molecular‐weight DNA to construct physical genome maps. These maps can be used to reveal large structural variants, but can also be combined with sequencing assemblies to produce hybrid scaffolds of unprecedented lengths, with some spanning chromosomal ar In addition, when one aligns the sequence and BioNano assemblies, one can identify chimeric joins errors, which would appear as conflicting alignment junctions. Chimeric joins – two distal regions in the genome are incorrectly placed together by assembly algorithms may form when short reads, molecules, or paired‐end inserts are unable to span across long DNA repeats. We developed a new hybrid scaffold pipeline that detects and resolves these conflicting junctions between the sequence and the BioNano assemblies. At a conflicting junction, the pipeline uses BioNano’s long molecules to determine which assembly has been constructed incorrectly. Specifically, it checks the chimeric quality scores surrounding the conflict junction on the genome map for any evidence of a misassembly. This score indicates the percentage of BioNano molecules aligned 55 kb to the left and to the right of a locus. If the junction on the genome map has low scores (less than 35%), then the genome map support would be considered relatively weak; hence, the pipeline would cut the genome map at the conflict, thus resolving the conflict. Conversely, if the genome map has high chimeric quality scores, then the sequence contig would be cut. Importantly, this automatic conflict‐resolution function can be manually modified to enable users to have fine control in generating high quality and complete hybrid scaffolds. We applied this hybrid scaffold pipeline on a haploid human genome (CHM1). The genome has been sequenced and genome mapped, and the assemblies’ N50 values are 27.7 Mb and 3.9 Mb, respectively. The pipeline resolved 23 chimeric joins in the sequence assembly and three in the BioNano genome maps. Moreover, by combining the two refined assemblies, the ultra‐long hybrid scaffolds resulted in a 58.4 Mb N50 value and 2.9 Gb in length. This new hybrid scaffold functionality further enhances the construction of highly accurate and contiguous reference assemblies for complex plants and animal genomes using BioNano mapping technology. 125
11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting DE NOVO ASSEMBLY AND STRUCTURAL VARIATION DETECTION OF HUMAN GENOMES USING SINGLE MOLECULE NEXT-GENERATION MAPPING AND SV CALL VALIDATION BY INHERITANCE AND ORTHOGONAL MEASUREMENTS. Thursday, 2nd June 17:35 La Fonda Ballroom Tech Talk (TT‐2.09) Alex Hastie 1 , Thomas Anantharaman 1 , Tiffany Liang 1 , Ernest Lam 1 , Joyce Lee 1 , Khoa Pham 1 , Michael Saghbini 1 , Ali Bashir 2 , Han Cao 1 1 BioNano Genomics, 2 Mount Sinai School of Medicine Structural variation detection is generally based on reference mapping to find discordant evidence. In order to detect structural variations comprehensively, a reference independent (de novo) approach is needed as it allows assembly of regions absent from the reference. Using Next‐Generation Mapping (NGM) from BioNano Genomics), we produced high‐resolution genome maps that were de novo assembled and preserved the long‐range genomic information necessary for structural variation detection. Here, the Genome in a Bottle (GIAB) reference trio of Ashkenazi Jewish descent (NA24385, NA24149, NA24143) has been de novo assembled using the Irys System. Structural variation analysis reveals insertions, inversions, and deletions, including large deletions in the UGT2B17 gene (involved in graft versus host disease, osteopathic health and testosterone and estradiol levels) in the mother and son. We compared structural variants found in the son (NA24385) by genome mapping to those found in his parents. Deletion and insertion calls, one kilobase and up, found in the son are also found in the parents at a rate of 90% and 92% respectively. We also use structural variation calls made with PacBio sequencing by reference mapping and local assembly approaches to validate BioNano’s structural variation calls, resulting in up to 80% cross‐validation rates for deletions of 2‐5kb, while a much lower rate for larger deletions and all insertions by PacBio’s methods. When considering PacBio SV calls, BioNano calls could validate all categories at a rate of 80‐90%. Thus, structural variation calling by next generation mapping is a fast, inexpensive, robust and accurate method that can be orthogonally validated in size bins that other methodologies are able to interrogate as well as provide novel SV information where other technologies are unable to interrogate. 126
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Sequencing

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