Incorporating sequence capture into library preparation for MinIONTM, GridIONTM and PromethIONTM Hybrid sequence capture allows users to select thousands of loci of interest simultaneously prior to sequencing, making more efficient use of the sequencing run Contact: [email protected] More information at: www.nanoporetech.com and publications.nanoporetech.com gDNA a) Randomly fragmented ~6 kb library b) Human exome report containing regions of interest Fragmentation OVERVIEW PER GENE OVERVIEW PER GENE Reads by exit status Alignments per gene End-prep Showing the exit status of all the reads analysed Select a gene in the table below to show summary Size-selection and p A p USP17L8 hybridisation to A Filter . NCBI gene ID 100287144 Ensembl: ENSG00000231396 T Barcoded PCR- biotinylated capture probes Ubiquitin-specific peptidase 17-like family member 10 adapter ligation Gene Estimated coverage Alignment count B B B B B B B B B B B B B B B B B B 82.8% B B B B USP17L8 114 X 350 B 105 81 X 86.6% B successful Alignments Estimated coverage Average accuracy B B B B B B B B B B B B B B USP17L7 87 X 127 B B B B B B B B B B B B B B B B F PCR with R universal primers USP17L10 81 X 105 100 Coverage Gene coverage Capture of probe- USP17L12 73 X 90 50 Alignment quality too low 96,424 reads template duplexes POM121L7 67 X 115 Workflow successful onto beads 465,170 reads CTAGE9 62 X 90 0 0 500 1,000 1,500 2,000 Reference sequence position B B B B B B B B B CTAGE1 61 X 148 B B B B B B B B B Pooling and sequence capture B B B B B B B B B FAM90A26 58 X 197 B B B B B B B B B 70 Count Accuracy distribution Alignments - key figures MST1 58 X 190 35 Elution and PCR TSPY2 57 X 94 Elution, amplification, 0 adapter-ligation 80 85 90 95 465,170 17,702 86.6% First < 1 of 1768 > Last and sequencing Amplicon-sequencing protocol Alignments Genes Average accuracy Accuracy (%) Fig. 1 Long-read sequence capture a) workflow overview b) multiplexed sequence capture Fig. 2 Analysis report of sequence-capture data for the human exome Sequence capture uses complementary Resequencing analysis workflow for probes to enrich for targets of interest sequence-capture experiments Sequence capture is a technique which allows the enrichment of specific regions of interest from We have released an updated analysis workflow for sequence-capture experiments. To illustrate a genome. It is useful when: this, we captured the human exome using Agilent’s SureSelect Human All Exon V6 panel, and i) the user is not interested in analysing the entire genome generated ~ 2.35 Gb of 1D sequence data from a MinION run, representing ~40x average ii) the genome is too large for the throughput of the sequencer coverage. We analysed the data using the resequencing analysis workflow (Fig. 2). Following iii) the user wishes to save money and time on sequencing and analysis iv) the regions are longer than can be amplified by PCR, or too many PCRs would be required. basecalling, reads are mapped to the human exome reference sequence. Individual gene information is displayed, including coverage and read-accuracy distribution at that position. Sequence capture is performed during library preparation by hybridising the library fragments to Future releases of this application will support the uploading of target regions, highlight known probes which are specific to the regions of interest (Fig. 1). SNPs in the target regions and allow those SNPs to be displayed with a confidence value. a) BRCA1, position: chr17:41569479–41650296, band: 17q21.31 Chromosome 1: 16,536,698–16,639,185 p31.3 p31.3 q32.1 q41 q43 q44 p36.13 80,818 bp Forward strand Contig 1.00 Mb < EPHA2 RP4-733M16.2 > SZRD1 > NECAP2 > RP4-798A10.7 > RP5-1182A14.5 > < RP5-1182A14.6 < RP11-108M9.1 < MFAP2 RP11-276H7.2 > < MTL1 < RP11-430L17.1RP4-798A10.2 > < RP5-875O13.6MST1P2 RP1-163M9.7> > RP11-108M9.2 > RP1-37C10.3 > RP11-276H7.3 > < FBXO42 < SPATA21 < CROCCP3 < RP5-875O13.1< CROCCP2< RP5-875O13.7 RP11-108M9.3 > < ATP13A2 < ARHGEF19 RP4-798A10.4 > RP1-163M9.8 > < RP5-1182A14.7 < RP11-108M9.4 < SDHB b) Tyr1563Ter, wild type is C and SNP is G < ANO7P1 FAM231B > FAM231.A > CEOCC > RP11-108M9.5 > < RSG1 < EIF1AP1RP1-163M9.5 > < RP11-108M9.6 A T T C C T T T C A G A G G G A A * C C C T T A C C T G G A A T C T G G A A T C A G C C T C T T C T C T G A < ESPNP < MST1L A T T C C T T T G A G A G G G A A C C C C T T A C C T G G A A T C T G G C A T C A G C C T C T T C T C T G A < NBPF1 A T T C C A T T C A G A G G G A A C C C C T T A G C T G G A A T C T G G A A T C A G C C T C T T C T C T G A A T T C C T T T C A G A G G G A A C C C C T T A G C T G G A A T C T G G A A T C A G C C T C G T C T C T G A A T T C C T T T C A G A G G G A A C C C C T T A C C T G G A A T C T T G A A T C A G C C T C T T C T C T G A A T T C C T T T C A G A A G G A A C C C C T T A C C T G G A A T C T G G A A T C A G C C T C T T C T C T G A A T T C C T T T C A G A G G G A A C C C C T T A G C T G G A A T C T G G A A T C A G C C T C T T C A C T G A A T T C C T T T C A G A G G G A A C C C C T T A C C T G G A A T C T G G A A A C A G C C T C T T C T C T G A A T T C C T T T C A G A G G G A A C C C C T T A G C T G G * A T C T G G A A T C A C C C T C T T C T C T G A 40 kb A T T C C T T T C A G A G G G * A C C C C T T A C C T G G A A T C T G G A A T C A G C C T C T T C T C T G A A * T C C T T T C A G A G G G A A C C C C T T A G C T G G A A T C T G G A A T C A G C C T C T T C T C T G A A T T C C T T T C A G A G G G A A T C C C T T A G C T G G A A T C T G G A A T T A G C C T C T T C T C T G A A T T C C T T T C A G A G G G A A C C C C T T A C C T G G A A T C T G G A A T C A G C C T C T T C T C T G A A T T C C T T T C A G A G G G A A C C C C T T A C C T G G A A T C T G G A A T C A G C C T C T T C T C T T A A T T C C T T T * A G A G G G A A C C C C T T A G C T G G A A T C T G G A A T C A G C C T C T T G T C T G A A T T C C T T T C A G A G G G A A C C C C T T A C C T G G A A T C T G G A A T C A G C C T C T T C T C T G A A T T C C T T T C * G A G G G A A C C C C T T A C C T G A A A T C T G G A A T C A G C C T C T T C T C T G A A T T C C T T T C A G C G G G A A C C C C T T A G C T G G A A T C T G G * A T C A G C C T C T T C T C T G A c) Arg1443Gly, wild type is C and SNP is G A A G T G A C T C T T C T G C C C T T G A G G A C C T G G G A A A T C C A G A A C A A A G C A C A T C A G A A A G T G A C T C T T C T G C C C T T G A G G A C C T G C G A A A T C * A G A A C A A A G C A C A T C A G A A A G T G A C T * C T C T G C C C T T G A G G A C C T G G G A A A T C C A G A A C A A A G C A C G T C A G A A A G T G A C T C T T C T G C C C T T G A G G A C C T G G G A A A T C C A G A A C A A A G C A C A T C A G A T A G T G A C T C T T C T G C C C T T G A G G A C C T G G G A A A T C C A G A A C A A A G C A C A T C A G A A A G T G A C T C T * * * G C C C T T G A G G A C C T G C G A A A T C C A G A A C A A A G C A C A T C A G A A A G T G A C T C T T C T * C C C T T G A G G A C C T G C G A A A T C C A G A A C A A A G C A C A T C A G A A A G T G * C T C T T C T G C C C T T G A G G A C C T G G G A A A T G C A G A A C A A A G C A C A T C A G A A A G T G A C T C C T C T G C C C T T G A G G A C C T G C G A A A T C C A G A A C A A A G C A C A T C A G A A A G T G A C T C T A C T G C C C T T G A G G A C C T G C G A A A T C C A G A A C A A A G C A A A T C A G A A A G T G A C T C T T C T G C C C T T G A G G A C C T G G G A A A T C C A G A A C A A A G C A C A T C A G A Fig.