1. Sample & Assay Technologies
Next Generation Sequencing:
An introduction to applications and technologies
Quan Peng, Ph.D.
Scientist, R&D
Quan.Peng@QIAGEN.com

2. 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, 2013
Speaker:
Quan Peng, Ph.D.
Webinar 2:
Date:
Speaker:
Next-generation sequencing for cancer research
April 11, 2013
Vikram Devgan, Ph.D., MBA
Webinar 3:
Date:
Speaker:
Next-generation sequencing data analysis for genetic profiling
April 18, 2013
Ravi Vijaya Satya, Ph.D.
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3. Sample & Assay Technologies
Agenda
Next Generation Sequencing
Background
Technologies
Applications
Workflow
Targeted Enrichment
Methodology
Data analysis
New product released!
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4. Sample & Assay Technologies
DNA Sequencing – The Past Decade
Output (Kb)
10E+8
10E+6
10E+4
10E+2
Adapted from ER Mardis. Nature 470, 198-203 (2011) doi:10.1038/nature09796
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Rapid Decrease in Cost
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What is Next-Generation Sequencing?
Sanger Sequencing
DNA is fragmented
NGS: Massive Parallel Sequencing
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)
Direct step-by-step detection of
each nucleotide base
incorporated
during the sequencing reaction
.
Separation by electrophoresis
Readout with fluorescent tags
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7. 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
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8. 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
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9. Sample & Assay Technologies
Single-end
reading
Single-end vs. paired-end reading
2nd 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
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10. 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
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11. 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
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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)
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NGS Applications
Next Generation Sequencing
Genomics
Transcriptomics
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Epigenomics
Metagenomics
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NGS Applications
Next Generation Sequencing
Genomics
Transcriptomics
Epigenomics
Metagenomics
DNA-Seq
Mutation,
SNVs,
Indels,
CNVs,
Translocation
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15. Sample & Assay Technologies
NGS Applications
Next Generation Sequencing
Genomics
Transcriptomics
DNA-Seq
RNA-Seq
Mutation,
SNVs,
Indels,
CNVs,
Translocation
Expression level,
Novel transcripts,
Fusion transcript,
Splice variants
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Epigenomics
Metagenomics
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16. Sample & Assay Technologies
NGS Applications
Next Generation Sequencing
Genomics
Transcriptomics
Epigenomics
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
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Metagenomics
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NGS Applications
Next Generation Sequencing
Genomics
Transcriptomics
Epigenomics
Metagenomics
DNA-Seq
RNA-Seq
ChIP-Seq,
Methyl-Seq
MicrobialSeq
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,
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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
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19. Sample & Assay Technologies
QIAGEN’s Solution for NGS Workflow
Sample
preparation
Target Enrichment kit
HMW DNA prep kit
Single Cell/WGA kit
Library
construction
rRNA depletion kit
ChIP-seq Kit
Pathogen bacteria prep kit
Library construction kit
MinElute size selection kit
Library quantification kit
Sequencing
Data
analysis
Result
validation
GeneRead DNAseq data
analysis web portal
RT2 Profiler PCR Arrays
Somatic Mutation PCR Arrays
Pyrosequencing
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CNA/CNV PCR Arrays
EpiTect ChIP PCR Arrays
SNP PCR Arrays
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20. 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
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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
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Hybridization
capture
(24-72 hrs)
Sequencing
Data analysis
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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)
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Library
construction
Sequencing
Data analysis
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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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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
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25. Sample & Assay Technologies
GeneRead DNAseq Custom Panel
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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
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27. Sample & Assay Technologies
NGS Data Analysis
Reference sequence
A
alignment
Sequencing reads
C
C
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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 0.001 – 0.01%)
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
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coverage depth = 3
coverage depth = 2
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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
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On-target
reads
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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 = 3
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coverage depth = 2
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31. Sample & Assay Technologies
QIAGEN’s Solution
FREE Complete & Easy to use Data Analysis with Web-based Software
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Summary
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
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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. Sample & Assay Technologies
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: 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
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