Sequencing

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11th Annual Sequencing, Finishing, and Analysis in the Future Meeting ANALYSIS OF MITOCHONDRIAL GENOME ISOFORMS IN LETTUCE Wednesday, 1st June 20:00 La Fonda Mezzanine (2nd Floor) Poster (PS‐2b.09) Alexander Kozik, Jie Li, Dean Lavelle, Sebastian Reyes Chin Wo, Richard Michelmore Genome Center, University of California, Davis, CA Mitochondria are integral organelles of most eukaryotic cells that have distinct genomes from the main nuclear one. Plant mitochondrial genomes are much larger than mitochondrial genomes of animals and fungi. Frequently, plant mitochondrial genomes also possess fragments of chloroplast genomes. Interchange between mitochondrial and nuclear genome segments are common phenomena in plants. Moreover, mitochondrial genome of a particular plant species may consist of several structural isoforms that coexist simultaneously. These isoforms are represented by the shuffling of large basic DNA sequence blocks separated by short repeats. The complexity of plant mitochondrial genomes consequently makes determining their sequence and structure challenging. There are fewer annotated plant mitochondrial genomes at NCBI GenBank than completed animal mitochondrial sequences. Only approximately 100 mitochondrial genomes are available for vascular plant species http://www.ncbi.nlm.nih.gov/genome/browse/?report=5. As a part of Lettuce Genome Sequencing Project https://lgr.genomecenter.ucdavis.edu/ we have assembled and characterized the lettuce mitochondrial genome. We extracted mitochondrial components from the Illumina and PacBio libraries used to assemble the lettuce nuclear genome and assembled them into a complete mitochondrial genome using a multistep approach. Initial assembly with CLC Genomics Workbench, Velvet, and FALCON generated multiple contigs that appeared to be the basic building blocks of several isoforms. The contact frequency for contig terminals with Illumina mate libraries, and contig bridging with PacBio reads were used to assemble the contigs into circular graphs. The total unique sequence of the lettuce mitochondrial genome is ~ 300 Kb. Multiple circular isoforms of the lettuce mitochondrial genome were identified and annotated. 97
11th Annual Sequencing, Finishing, and Analysis in the Future Meeting THE EXOME COVERAGE AND IDENTIFICATION (EXCID) REPORT: A GENE SURVEY TOOL FOR CLINICAL SEQUENCING APPLICATIONS Wednesday, 1st June 20:00 La Fonda Mezzanine (2nd Floor) Poster (PS‐2b.10) Rashesh Sanghvi, Kimberly Walker, Qiaoyan Wang, Harsha Doddapaneni, Yi Han, Huyen Dinh, Eric Boerwinkle, Donna Muzny, Richard Gibbs Baylor College of Medicine The Exome Coverage and Identification (ExCID) Report was developed at the Baylor College of Medicine Human Genome Sequencing Center (BCM‐HGSC) to represent gene, transcript and exon sequence coverage for samples analyzed with panel sequencing, WES and WGS. Since March 2013, the report has been used to analyze more than 22,888 WES, 9,428 regional captures and 32,660 WGS research samples for the BCM‐HGSC and more than 23,647 WES/Carrier/CAP‐CLIA samples at Baylor Miraca Genetics Laboratories (BMGL). ExCID is a one‐stop tool for providing annotations, gathering mapping metrics such as %reads mapped and %duplicates reads, coverage metrics such as average coverage and bases 20X, gene% coverages for OMIM genes and reporting exact bases that are poorly covered based on user‐defined coverage threshold. ExCID provides gene, transcript and exon annotations to the input BED regions from RefSeq, VEGA, CCDS and MIRbase. These external databases are downloaded with the installation of ExCID and can be updated using the scripts provided in the installation. The user can visualize coverage over the region of interest using the UCSC tracks (WGS and BigBed) and the OMIM Gene % coverages bar chart. Results are reported as boxplots and other ‘coverage tracks’ that can be visualized in popular browsers such as the Integrative Genome Viewer (IGV) and the UCSC Genome Browser. ExCID output was utilized by BMGL website to provide clinicians easy access to in‐depth information on clinical design aspects and coverage. In addition, ExCID’s batch ability makes it possible to compare data from hundreds of samples to reveal trends in the performance of large scale sequencing projects and asses the consistency of sequencing strategies. This can be used to determine the common problematic regions across samples leading to design improvements and unique solutions. Analyzing clinical WES samples led to technical developments such as capture reagent spike‐in (i.e. panel killer) and improving coverage of nearly all clinically targeted genes (N=3,200 at 100% coverage). Furthermore, ExCID is used for evaluating the efficiency of in‐house pipelines to meet requirements and set thresholds for various coverage metrics. ExCID’s ability to asses the percentage of gene covered is routinely used both clinical and research setting to samples with irregular performance. ExCID has also been used by the Clinical Sequencing Exploratory Research (CSER) Sequencing Standards working group, a consortium of ten clinical sequencing centers across the country, to determine the regions of the genome that remain difficult to sequence, regardless of the method used. Establishment of robust WGS pipeline metrics are essential for broad applications in both the research and clinical setting. 98
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Reliable solutions for focused NGS
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Sequencing

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