Next Generation Sequencing: An introduction to applications and technologies

Transcription

1 Next Generation Sequencing: An introduction to applications and technologies Quan Peng, Ph.D. Scientist, R&D

2 Welcome to the three-part webinar series Next Generation Sequencing and its role in cancer biology Webinar 1: Next-generation sequencing, an introduction to technology and applications Date: March April 4, 2013 Speaker: Quan Peng, Ph.D. Webinar 2: Next-generation sequencing for cancer research Date: April 11, 2013 Speaker: Vikram Devgan, Ph.D., MBA Webinar 3: Next-generation sequencing data analysis for genetic profiling Date: April 18, 2013 Speaker: Ravi Vijaya Satya, Ph.D. Title, Location, Date 2

3 Agenda Next Generation Sequencing Background Technologies Applications Workflow Targeted Enrichment Methodology Data analysis New product released! 3

4 DNA Sequencing The Past Decade 10E+8 Output (Kb) 10E+6 10E+4 10E+2 Adapted from ER Mardis. Nature 470, (2011) doi: /nature09796 Title, Location, Date 4

5 Rapid Decrease in Cost Title, Location, Date 5

6 What is Next-Generation Sequencing? Sanger Sequencing NGS: Massive Parallel Sequencing DNA is fragmented.dna is fragmented Adaptors ligated to fragments (Library construction) Cloned to a plasmid vector Cyclic sequencing reaction Clonal amplification of fragments on a solid surface (Bridge PCR or Emulsion PCR) Separation by electrophoresis Readout with fluorescent tags.direct step-by-step detection of each nucleotide base incorporated during the sequencing reaction Title, Location, Date 6

7 Bridge PCR DNA fragments are flanked with adaptors (Library) A flat surface (chip) coated with two types of primers, corresponding to the adaptors Amplification proceeds in cycles, with one end of each bridge tethered to the surface Clusters of DNA molecules are generated on the chip. Each cluster is originated from a single DNA fragment Used by Illumina Title, Location, Date 7

8 Illumina HiSeq/MiSeq Run time 1-10 days Produces Gb of sequence Read length 2X100 bp 2X250bp (pair end) Cost: $ $0.4/Mb Title, Location, Date 8

9 Single-end vs. paired-end reading Single-end reading 2 nd strand synthesis Pair-end reading Single-end reading (SE): Sequencer reads a fragment from only one end to the other Pair-end reading (PE): Sequencer reads both ends of the same fragment More sequencing information, reads can be more accurately placed ( mapped ) May not be required for all experiments, more expensive and time-consuming Title, Location, Date 9

10 Emulsion PCR Fragments, with adaptors, are PCR amplified within a water drop in oil One primer is attached to the surface of a bead DNA molecules are synthesized on the beads. Each bead bears DNA originated from a single DNA fragment Beads with DNA are then deposit into the wells of sequencing chips, one well one bead Used by Roche 454, IonTorrent and SOLiD Title, Location, Date 10

11 Ion PGM/ Proton Run time 3 hrs Read length bp; homopolymer can be an issue Throughput determined by chip size (ph meter array): 10Mb 5 Gb Cost: $1 - $20/Mb Title, Location, Date 11

12 Multiplex Sequencing Barcoding Samples Depending on the application, we may not need to generate so many reads per sample Multiple samples with different index can be combined and put into one sequencing run or into one sequencing lane Save money on sequencing costs (pay per sample) Title, Location, Date 12

13 NGS Applications Next Generation Sequencing Genomics Transcriptomics Epigenomics Metagenomics Title, Location, Date 13

14 NGS Applications Next Generation Sequencing Genomics Transcriptomics Epigenomics Metagenomics DNA-Seq Mutation, SNVs, Indels, CNVs, Translocation Title, Location, Date 14

15 NGS Applications Next Generation Sequencing Genomics Transcriptomics Epigenomics Metagenomics DNA-Seq RNA-Seq Mutation, SNVs, Indels, CNVs, Translocation Expression level, Novel transcripts, Fusion transcript, Splice variants Title, Location, Date 15

16 NGS Applications Next Generation Sequencing Genomics Transcriptomics Epigenomics Metagenomics DNA-Seq RNA-Seq ChIP-Seq, Methyl-Seq Mutation, SNVs, Indels, CNVs, Translocation Expression level, Novel transcripts, Fusion transcript, Splice variants Global mapping of DNA-protein interactions, DNA methylation, histone modification Title, Location, Date 16

17 NGS Applications Next Generation Sequencing Genomics Transcriptomics Epigenomics Metagenomics DNA-Seq RNA-Seq ChIP-Seq, Methyl-Seq Microbial- Seq Mutation, SNVs, Indels, CNVs, Translocation Expression level, Novel transcripts, Fusion transcript, Splice variants Global mapping of DNA-protein interactions, DNA methylation, histone modification Microbial genome Sequence, Microbial ID, Microbiome Sequencing, Title, Location, Date 17

18 Next Generation Sequencing Workflow Sample preparation Isolate samples (DNA/RNA) Qualify and quantify samples Several hours to days Library construction Prepare platform specific library Qualify and quantify library 4-8 hours Sequencing Perform sequencing run reaction on NGS platform 8 hours to several days Data analysis Application specific data analysis pipeline Several hours to days Title, Location, Date 18

19 QIAGEN s Solution for NGS Workflow Sample preparation Target Enrichment kit HMW DNA prep kit Single Cell/WGA kit rrna depletion kit ChIP-seq Kit Pathogen bacteria prep kit Library construction Library construction kit MinElute size selection kit Library quantification kit Sequencing Data analysis GeneRead DNAseq data analysis web portal Result validation RT 2 Profiler PCR Arrays Somatic Mutation PCR Arrays Pyrosequencing CNA/CNV PCR Arrays EpiTect ChIP PCR Arrays SNP PCR Arrays Title, Location, Date 19

20 GeneRead DNAseq Gene Panel: Targeted Sequencing What is targeted sequencing? Sequencing a sub set of region in the whole-genome Why do we need targeted sequencing? Not all regions in the genome are of interest or relevant to specific study Exome Sequencing: sequencing most of the coding regions of the genome (exome). Protein-coding regions constitute less than 2% of the entire genome Focused panel/hot spot sequencing: focused on the genes or regions of interest What are the advantages of focused panel sequencing? More coverage per sample, more sensitive mutation detection More samples per run, lower cost per sample Title, Location, Date 20

21 Target Enrichment - Methodology Hybridization capture Large DNA input (1 ug) Long processing time (2-3 days) Large throughput (MB region to whole exome) Sample preparation (DNA isolation) Library construction Hybridization capture (24-72 hrs) Sequencing Data analysis Title, Location, Date 21

22 Target Enrichment - Methodology Multiplex PCR Small DNA input (< 100ng) Short processing time (several hrs) Relatively small throughput (KB - MB region) Sample preparation (DNA isolation) PCR target enrichment (2 hours) Library construction Sequencing Data analysis Title, Location, Date 22

23 GeneRead DNAseq Gene Panel Multiplex PCR technology based targeted enrichment for DNA sequencing Cover all human exons (coding region + UTR) Division of gene primers sets into 4 tubes; up to 1200 plex in each tube 23

24 GeneRead DNAseq Gene Panel Focus on your Disease of Interest Comprehensive Cancer Panel (124 genes) Disease Focused Gene Panels (20 genes) Breast cancer Colon Cancer Gastric cancer Leukemia Liver cancer Genes Involved in Disease Lung Cancer Ovarian Cancer Prostate Cancer Genes with High Relevance 24

25 GeneRead DNAseq Custom Panel 25

26 NGS Data Analysis Base calling From raw data to DNA sequences, generate sequencing reads Mapping to a reference Align the reads to reference sequences Can be considered as blast millions of sequences against reference database Variants identification Identify the differences between sample DNA and reference DNA Variant prioritization/filtering/validation Title, Location, Date 26

27 NGS Data Analysis Reference sequence A Sequencing reads alignment C C Title, Location, Date 27

28 NGS Data Analysis: Sequencing Depth Coverage depth (or depth of coverage): how many times each base has been sequenced or read Unlike Sanger sequencing, in which each sample is sequenced 1-3 times to be confident of its nucleotide identity, NGS generally needs to cover each position many times to make a confident base call, due to relative high error rate (0.1-1% vs %) Increasing coverage depth is also helpful to identify low frequent mutation in heterogenous samples such as cancer sample Reference sequence NGS reads coverage depth = 4 coverage depth = 2 coverage depth = 3 Title, Location, Date 28

29 NGS Data Analysis: Specificity Specificity: the percentage of sequences that map to the intended targets region of interest number of on-target reads / total number of reads Reference sequence ROI 1 ROI 2 NGS reads Off-target reads On-target reads On-target reads Title, Location, Date 29

30 NGS Data Analysis: Uniformity Reference sequence Coverage uniformity: measure the evenness of the coverage depth of target position Calculate coverage depth of each position Calculate the median coverage depth Set the lower boundary of the coverage depth related to median depth (eg. 0.1 X median coverage depth) Calculate the percentage of target region covered by equal or more than the lower boundary NGS reads coverage depth = 10 coverage depth = 2 coverage depth = 3 Title, Location, Date 30

31 QIAGEN s Solution FREE Complete & Easy to use Data Analysis with Web-based Software 31

32 Summary Run Summary Specificity Coverage Uniformity Numbers of SNPs and Indels Summary By Gene Specificity Coverage Uniformity # of SNPs and Indels 32

33 Features of Variant Report SNP detection Indel detection 33

34 QIAGEN s GeneRead DNAseq Gene Panel System FOCUS ON YOUR RELEVANT GENES Focused: Biologically relevant content selection enables deep sequencing on relevant genes and identification of rare mutations Flexible: Mix and match any gene of interest NGS platform independent: Functionally validated for PGM, MiSeq/HiSeq Integrated controls: Enabling quality control of prepared library before sequencing Free, complete and easy of use data analysis tool

35 Upcoming webinars Next Generation Sequencing and its role in cancer biology Webinar 2: Next-generation sequencing for cancer research Date: April 11, 2013 Speaker: Vikram Devgan, Ph.D., MBA Register here: Webinar 3: Next-generation sequencing data analysis for genetic profiling Date: April 18, 2013 Speaker: Ravi Vijaya Satya, Ph.D. Register here: ps://www2.gotomeeting.com/register/ Title, Location, Date 35