DNA Sequencings

The Genome Access Course
Next Generation DNA Sequencing
Illumina HiSeq X
1.8 Tbp
(3 billion reads) in ~3 days
(as of 11/6/2014)
November 2014
Whole Genome Shotgun Sequencing
Randomly
Fragment
Genomic DNA
Sequence
Fragments
Genome Assembly ...ATCCGTAAATGGGCTGATACTACTAATGC

TGGGCTGATACTACTAATGCCAAACTGTACTAGTCCTG...
...ATCCGTAAATGGGCTGATACTACTAATGCCAAACTGTACTAGTCCTG...
Contiguous
Sequence
(Contig)
November 2014
RNA Sequencing (RNA-Seq)

cDNA made
from RNA
Sequence
cDNA
Fragments
Garber et al, Nat Methods (2011)
1. Characterize all RNA in sample
2. Gene expression level proportional to number of reads
3. Detect alternatively spliced transcripts

November 2014
Typical Next Gen Experiments
• Genome sequencing
– Novel genomes
– Resequencing
• Transcriptome sequencing (RNA-seq)
– Characterize transcripts with or without reference genome
• Typical length
• Short (microRNAs, …)
– Find differentially expressed transcripts
• Other
– Methyl-seq
– ChIP-seq
November 2014
November 2014
Illumina Sequencing
Sequencing by
DNA Sample Synthesis
Construct
Library
Cluster Generation
in Flow Cell
200+ million reads per lane

(>100 bp reads)
November 2014
Types of Sequencing Libraries

Single-End Reads - 5’ or 3’ (random)
Paired-End Reads - 5’ and 3’
200-500 bp
Mate-Pair Reads - 5’ and 3’
2-5 kbp
November 2014
Taken from GIGA Newsletter 13 – Universite de Liège
November 2014
What Does the Data Look Like?

FASTQ File Format
Sequence
Quality
(ASCII character for
each base)
> 200 million

reads in one
lane
Files so big
that they
break them
up in 40
million reads
per file
November 2014
Example Analysis Workflow

Paired-End FASTQ Files
FASTQ FASTQ
(_R1.txt) (_R2.txt)
Align Reads to Genome
FastQC SAM File

(Diagnostics)
Trim Reads BAM File

(if needed)
November 2014
Sequence Composition Diagnostics
Unbiased Reads
Biased Reads
First Position Nearly

Always “T”
November 2014
GC Bias in First ~15 bp Due to Random Hexamer Priming
November 2014
Trim Sequences Prior To Analysis

• Make sure sequencing adapters are removed
• Trim ends of sequence based on quality scores
November 2014
FastX Toolkit – Hannon Lab at CSHL
Trimmomatic
November 2014
Example Analysis Workflow

Paired-End FASTQ Files
FASTQ FASTQ
(_R1.txt) (_R2.txt)
Align Reads to Genome
FastQC SAM File

(Diagnostics)
Trim Reads BAM File

(if needed)
November 2014
Sequence Alignment/Map (SAM) Format
Common file format to store:

- Reads
- Quality of each base
- How reads align to a reference sequence
Generated by most next gen analysis software
samtools software package
November 2014
samtools Used to Manipulate SAM Files
SAM File
samtools
PileUp
File BAM File
Call
Variants
…
Pileup output file
chr1 272 T 24 ,.$.....,,.,.,...,,,.,..^+. <<<+;<<<<<<<<<<<=<;<;7<&
chr1 273 T 23 ,.....,,.,.,...,,,.,..A <<<;<<<<<<<<<3<=<<<;<<+
chr1 274 T 23 ,.$....,,.,.,...,,,.,... 7<7;<;<<<<<<<<<=<;<;<<6
chr1 275 A 23 ,$....,,.,.,...,,,.,...^l. <+;9*<<<<<<<<<=<<:;<<<<
chr1 276 G 22 TTTTTTTTTTTTTTTTTTTTTTT 33;+<<7=7<<7<&<<1;<<6<
chr1 277 T 22 ....,,.,.,.C.,,,.,..G. +7<;<<<<<<<&<=<<:;<<&<
chr1 278 G 23 ....,,.,.,...,,,.,....^k. %38*<<;<7<<7<=<<<;<<<<<
chr1 279 C 23 A..T,,.,.,...,,,.,..... ;75&<<<<<<<<<=<<<9<<:<<
November 2014
Binary Alignment (BAM) Files
• Common file format to store reads and their alignment to a

reference sequence
– Generated by most next gen analysis software
• samtools software package
• UCSC Genome Browser and Ensembl can display them as a

custom track
– IGV from Broad very useful
November 2014
UCSC Genome Browser with

1,000 Genomes Project Data
November 2014
Integrated Genomics Viewer (IGV)
November 2014
LookSeq at Sanger Mouse Genomes Project
November 2014
Glo1 CNV Present in Mouse Genomes Data for A/J

Proximal Flank Glo1 Locus Distal Flank
Chr17: 30.5Mb Chr17: 30.7Mb Chr17: 31.2Mb
Max ~50x coverage Max >100x coverage Max ~50x coverage
50kb 50kb 50kb
November 2014
Glo1 CNV Not Present in Mouse Genomes Data for NZO

Proximal Flank Glo1 Locus Distal Flank
Chr17: 30.5Mb Chr17: 30.7Mb Chr17: 31.2Mb
Max ~25x coverage Max ~25x coverage Max ~25x coverage
50kb 50kb 50kb
November 2014
Galaxy (http://main.g2.bx.psu.edu)
November 2014
Public Data Repositories

NCBI EBI
SRA Formatted
Files FASTQ Files
Automatically
Forward FASTQ
SRA ToolKit Files to Galaxy
FASTQ Files
November 2014
NCBI BioProject
November 2014
NCBI Gene Expression Omnibus
November 2014
Overall Analysis Workflow
FASTQ Files
Secondary Analysis Tertiary Analysis

1.Read Preprocessing & Diagnostics 1.Analysis of Read Counts
2.Align Reads to Reference e.g., Differentially expressed genes
2.Analysis of Gene Lists
1. Enrichment
2. Pathway and networks
3.Analysis of Aligned Reads 3.Analysis of Expression Patterns
e.g., Read counts per gene from RNA-Seq
November 2014
Push-Button Bioinformatics … Be Careful
November 2014
Third Generation Sequencing
November 2014

DNA Sequencings

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

DNA Sequencings

Uploaded by

Copyright:

Available Formats

The Genome Access Course

Next Generation DNA Sequencing

Genome Assembly ...ATCCGTAAATGGGCTGATACTACTAATGC

RNA Sequencing (RNA-Seq)

Garber et al, Nat Methods (2011)

1. Characterize all RNA in sample

2. Gene expression level proportional to number of reads

3. Detect alternatively spliced transcripts

Typical Next Gen Experiments

200+ million reads per lane

Types of Sequencing Libraries

Paired-End Reads - 5’ and 3’

Taken from GIGA Newsletter 13 – Universite de Liège

What Does the Data Look Like?

> 200 million

Example Analysis Workflow

FastQC SAM File

Trim Reads BAM File

Sequence Composition Diagnostics

First Position Nearly

GC Bias in First ~15 bp Due to Random Hexamer Priming

Trim Sequences Prior To Analysis

FastX Toolkit – Hannon Lab at CSHL

Example Analysis Workflow

FastQC SAM File

Trim Reads BAM File

Sequence Alignment/Map (SAM) Format

Common file format to store:

samtools Used to Manipulate SAM Files

Binary Alignment (BAM) Files

• Common file format to store reads and their alignment to a

• UCSC Genome Browser and Ensembl can display them as a

UCSC Genome Browser with

Integrated Genomics Viewer (IGV)

LookSeq at Sanger Mouse Genomes Project

Glo1 CNV Present in Mouse Genomes Data for A/J

50kb 50kb 50kb

Glo1 CNV Not Present in Mouse Genomes Data for NZO

50kb 50kb 50kb

Public Data Repositories

NCBI Gene Expression Omnibus

Overall Analysis Workflow

Secondary Analysis Tertiary Analysis

Push-Button Bioinformatics … Be Careful

Third Generation Sequencing

You might also like