Ngs part i 2013

Sample & Assay Technologies

Next Generation Sequencing: An introduction to applications and technologies

Quan Peng, Ph.D.Scientist, R&D

[email protected]

Sample & Assay Technologies 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, 2013Speaker: Quan Peng, Ph.D.

Webinar 2: Next-generation sequencing for cancer researchDate: April 11, 2013Speaker: 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

Sample & Assay Technologies Agenda

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� Next Generation Sequencing� Background� Technologies� Applications� Workflow

� Targeted Enrichment� Methodology� Data analysis� New product released!

Sample & Assay Technologies DNA Sequencing – The Past Decade


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b)

Adapted from ER Mardis. Nature 470, 198-203 (2011) doi:10.1038/nature09796

Sample & Assay Technologies Rapid Decrease in Cost


Sample & Assay Technologies What is Next-Generation Sequencing?


DNA is fragmented

Cloned to a plasmid vector

Cyclic sequencing reaction

Separation by electrophoresisReadout with fluorescent tags

Sanger Sequencing NGS: Massive Parallel Sequencing

.DNA is fragmented

Adaptors ligated to fragments(Library construction)

Clonal amplification of fragments on a solid surface(Bridge PCR or Emulsion PCR)

.Direct step-by-step detection of each nucleotide base incorporatedduring the sequencing reaction

Sample & Assay Technologies 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

Sample & Assay Technologies Illumina HiSeq/MiSeq


� Run time 1- 10 days

� Produces 2 - 600 Gb of sequence

� Read length 2X100 bp – 2X250bp (pair end)

� Cost: $0.05 - $0.4/Mb

Sample & Assay Technologies Single-end vs. paired-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

Single-endreading

2nd strandsynthesis

Pair-endreading

Sample & Assay Technologies 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

Sample & Assay Technologies Ion PGM/ Proton


� Run time 3 hrs

� Read length 100‐300 bp; homopolymer can be an issue

� Throughput determined by chip size (pH meter array): 10Mb – 5 Gb

� Cost: $1 - $20/Mb

Sample & Assay Technologies 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)

Sample & Assay Technologies NGS Applications


Next Generation Sequencing

Genomics Transcript-omics

Epi-genomics

Meta-genomics





Epi-genomics

Mutation, SNVs, Indels,CNVs,

Translocation

Meta-genomics

DNA-Seq





Epi-genomics


Translocation

Expression level,Novel transcripts, Fusion transcript,

Splice variants

Meta-genomics

DNA-Seq RNA-Seq





Epi-genomics


Translocation


Splice variants

Global mapping of DNA-protein

interactions, DNA methylation,

histone modification

Meta-genomics

DNA-Seq RNA-Seq ChIP-Seq, Methyl-Seq





Epi-genomics


Translocation


Splice variants

Global mapping of DNA-protein

interactions, DNA methylation,

histone modification

Meta-genomics

Microbial genomeSequence,

Microbial ID,MicrobiomeSequencing,

DNA-Seq RNA-Seq ChIP-Seq, Methyl-Seq

Microbial-Seq

Sample & Assay Technologies 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

Sample & Assay Technologies QIAGEN’s Solution for NGS Workflow


Sample preparation


Sequencing

Data analysis

� Target Enrichment kit� HMW DNA prep kit� Single Cell/WGA kit

� rRNA depletion kit� ChIP-seq Kit� Pathogen bacteria prep kit

� Library construction kit� MinElute size selection kit� Library quantification kit

Result validation

� GeneRead DNAseq data analysis web portal

� RT2 Profiler PCR Arrays� Somatic Mutation PCR Arrays� Pyrosequencing

� CNA/CNV PCR Arrays� EpiTect ChIP PCR Arrays� SNP PCR Arrays

Sample & Assay Technologies 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

Sample & Assay Technologies 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)


Hybridization capture

(24-72 hrs)Sequencing Data analysis

Sample & Assay Technologies 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

Sample & Assay Technologies 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

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Sample & Assay Technologies GeneRead DNAseq Gene Panel

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Genes Involved in Disease

Genes with High Relevance

� Comprehensive Cancer Panel (124 genes)

� Disease Focused Gene Panels (20 genes)

� Breast cancer

� Colon Cancer

� Gastric cancer

� Leukemia

� Liver cancer

� Lung Cancer

� Ovarian Cancer

� Prostate Cancer

Focus on your Disease of Interest


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GeneRead DNAseq Custom Panel

Sample & Assay Technologies 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

Sample & Assay Technologies NGS Data Analysis


Reference sequence

Sequencing reads

alignment

A

CC

Sample & Assay Technologies NGS Data Analysis: Sequencing Depth


Reference

sequence

NGS

reads

coverage depth = 4 coverage depth = 2coverage depth = 3

� 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 0.001 – 0.01%)

� Increasing coverage depth is also helpful to identify low frequent mutation in heterogenous samples such as cancer sample

Sample & Assay Technologies 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

NGS

reads

ROI 1 ROI 2

Off-target reads

On-target readsOn-target

reads

Sample & Assay Technologies NGS Data Analysis: Uniformity

� 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


Reference

sequence

NGS

reads

coverage depth = 10 coverage depth = 2coverage depth = 3

Sample & Assay Technologies QIAGEN’s Solution

� FREE Complete & Easy to use Data Analysis with Web-based Software

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Sample & Assay Technologies Summary

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Run Summary� Specificity� Coverage� Uniformity� Numbers of SNPs and Indels

Summary By Gene� Specificity� Coverage� Uniformity� # of SNPs and Indels


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Features of Variant Report

� SNP detection� Indel detection

Sample & Assay Technologies QIAGEN’s GeneRead DNAseq Gene Panel System

� 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

FOCUS ON YOUR RELEVANT GENES

Sample & Assay Technologies Upcoming webinars


Next Generation Sequencing and its role in cancer biology

Webinar 2: Next-generation sequencing for cancer researchDate: April 11, 2013Speaker: Vikram Devgan, Ph.D., MBARegister here: https://www2.gotomeeting.com/register/126404050

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/966970098

Ngs part i 2013

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