原标题：#生物工程#新一代测序技术NGS（CTGA2016 NIH NHGRI）全英干货 Next-Generation Sequencing Technologies Enjoy it! Next-generation Sequencing Technologies Massivelly Parallel sequencing basis NGS has transformed biomedical inquiry 1Library preparation 2 library plating 3 subclone DNA preparation 4 Subclone sequencing 5 eletrophoresis and detecton 1 genome fragmentation 2 end repair and adaptor ligation 3 surface attachment 4 amplification 5 sequencing Massively Parallel DNA sequencing instruments 1 all MPS platforms require a library obtained either by amplification or ligation with custom linkers (adapters) 2 Each library fragment is amplified on a solid surface (either bead or flat Si-derived surface) with covalently attached adapters that hybridize the library adapters 3 Direct step-by-step detection of the nucleotide base incorporated by each amplified library fragment set 4 A “digital”Read type that enables direct quantitative comparisons 5 shorter read lengths than capillary sequencers Library Construction for MPS 1 Shear high molecular weight DNA with sonication 2 Enzymatic treatments to blunt ends 3 Ligate synthetic DNA adapters(each with a DNA barcode), PCR amplify 4 Quantitate library 5 Proceed to WGS,or perform exome or specific gene hybrid capture PCR-related Problems in MPS 1 PCR is an effective vehicle for amplifying DNA, however.. 2 In MPS library construction, PCR can introduce preferential amplification of certain fragments 3 PCR also introduces false positive aretifacts due to substitution errors by the polymerase 4 Cluster formation is a type of PCR Hybrid Capture 1 Hybrid capture fragments from a whole genome library are selected by combining with probes that correspond to most human exons or gene targets. 2 The probe DNAs are biotinhlated, making selection from solution with streptavidin magnetic beads an effective means of purification. 3 An”exome” by definition, is the exons of all genes annotated in the reference genome. 4 Custom capture reagents can be synthesized to target specific loci that may be of clinical interest. Multiplex PCR Amplification of Targets 1 Design amplification primer pairs for exons of genes of interest; tile primers to overlap fragments in larger exons 2 Group primer pairs according to G+C content, Tm and reaction condition specifics 3 Amplify genomic DNA to generate multiple products from each primer set; pool products from each set 4 Create library by ligation or tail platform adaptors on the primer ends 5 Sequence Massively Parallel Sequencing by Synthesis Illumina Patterned Flow Cell ION Torrent-pH Sensing of Base Incorporation Post Data Generation Analyses Bioinformatic and computational approaches to NGS The Human Genome Reference enables MPS Genomics 1 The human genome reference sequence is the keystone interpreting MPS sequencing read data 2 Alignment of reads to the human reference sequence is the first step to identify variation of all types 3 Mis-aligning sequencs identify structural alterations 4 Alignment and analysis of RNA sequence data provides information about gene expression changes Single Nucleotide Variants; Insertion/deletions/ Structural Variants/ Copy Number Variations / Allele-specific expression/ Differentially Expressed Genes / Differentially Expressed Isoforms Reads are Aligned, Now What? 1 Data calibration and cleanup 2 Call SNPs 3 Evaluate Coverage 4 Analyze the data Tools Integrated Genomics Viewer RefCov: Coverage Depth and Breadth from Hybrid Capture Data and coverage characteristics False Negativity/Positivity 1 Most false negatives are due to lack of coverage 2 False positives are due to multiple reasons, including: Variant is only called on one strand Variant is only called at the end of the read Coverage of the matched normal at that locus is poor Gene has a pseudogene/paralog and the reads are mis-mapped High sensitivity variant calling algorithms have elevated false positive rates to achieve detection of subclonal variants and low false negative rates 3 Data that verifies or rufutes variant calls can help to define bioinformatic filters to remove them Third Generation Sequencers (Variations on a theme) Real Time Sequencing of Single DNA Molecules Pacific Bioscience PS 1 High molecular weight genomic DNA 2 Consistent shearing 30 kb 3 Sufficient DNA for PacBio sample prep The Human Reference is a work in Progress! 1 The current reference-GRCh38 is not optimal for some regions of the genome and/or some individuals/ ancestries. 2 GRCh 38 is comprised of DNA from several individual humans 3 Allelic diversity and structural variation present major challenges when assembling a representative diploid genome 4 New technologies, methods,and resources since 2003 have allowed for substantial improvements in the reference genome 5 Additional high-quality reference sequences are needed to represent the full range of genetic diversity in humans Improving the Human Reference Genomes Sequencing Plan First Glod Genome - NA 19240 Alignment of NA 19240 to BioNana map Finished BACs Resolve This Region Platinum/Glod Genome Status Oxford Nanopore Sequencing Genome-guided Immunotherapy Decision-making Identifying “non-self” Neoantigen Landscapes 1 NGS to identify tumor-specific antigens 2 Genome-guided Immunotherapy 3 pVac-Seq Pipeline: Open Source Neoantigen Prediction 4 Mutation/Neoantigen Load and Checkpoint Blockade Response Potential 5 Germline DNA Repair Defects and Checkpoint Blockade Reckade Response Potential GBM27: Rapid Progression in CNS GBM27:Clonal Evolution GBM27: Evolving Immune Respone 阅读原文为PPT **华盛顿大学当年承担了HGP 大约30%的人类基因组测序量返回搜狐，查看更多 责任编辑：