1. Next Generation DNA Sequencing
Illumina HiSeq X
1.8 Tbp
(3 billion reads) in ~3 days
(as of 11/6/2014)

2. Whole Genome Shotgun Sequencing
Randomly Fragment
Genomic DNA
Sequence
Fragments
Genome Assembly
...ATCCGTAAATGGGCTGATACTACTAATGC
TGGGCTGATACTACTAATGCCAAACTGTACTAGTCCTG...
...ATCCGTAAATGGGCTGATACTACTAATGCCAAACTGTACTAGTCCTG...
Contiguous Sequence (Contig)

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

4. 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

6. Illumina Sequencing Sequencing by Synthesis DNA Sample Construct
Library
Cluster Generation in Flow Cell
Batching effect per lane, per flow cell. Design your experiment to avoid batching samples in a particular sample group into one lane.
200+ million reads per lane
(>100 bp reads)

7. Types of Sequencing Libraries
Single-End Reads - 5’ or 3’ (random)
Paired-End Reads - 5’ and 3’ bp
Mate-Pair Reads - 5’ and 3’
Analysis software take parameters of how library was constructed into account.
2-5 kbp

8. dUTP replaces dUTT in second strand synthesis
Taken from GIGA Newsletter 13 – Universite de Liège

9. 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

10. Example Analysis Workflow
Paired-End FASTQ Files
FASTQ
(_R1.txt)
FASTQ
(_R2.txt)
Align Reads to Genome
FastQC
(Diagnostics)
SAM File
Trim Reads
(if needed)
BAM File

11. Sequence Composition Diagnostics
Unbiased Reads
Biased Reads
First Position Nearly Always “T” 11

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

13. Trim Sequences Prior To Analysis
Make sure sequencing adapters are removed
Trim ends of sequence based on quality scores

14. FastX Toolkit – Hannon Lab at CSHL
Trimmomatic

15. Example Analysis Workflow
Paired-End FASTQ Files
FASTQ
(_R1.txt)
FASTQ
(_R2.txt)
Align Reads to Genome
FastQC
(Diagnostics)
SAM File
Trim Reads
(if needed)
BAM File

16. 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

17. samtools Used to Manipulate SAM Files
BAM File
PileUp File
Call Variants …
Pileup output file
chr1 272 T 24 ,.$.....,,.,.,...,,,.,..^+. <<<+;<<<<<<<<<<<=<;<;7<&
chr1 273 T 23 ,.....,,.,.,...,,,.,..A <<<;<<<<<<<<<3<=<<<;<<+
chr1 274 T 23 ,.$....,,.,.,...,,,., <7;<;<<<<<<<<<=<;<;<<6
chr1 275 A 23 ,$....,,.,.,...,,,.,...^l. <+;9*<<<<<<<<<=<<:;<<<<
chr1 276 G 22 TTTTTTTTTTTTTTTTTTTTTTT 33;+<<7=7<<7<&<<1;<<6<
chr1 277 T ,,.,.,.C.,,,.,..G. +7<;<<<<<<<&<=<<:;<<&<
chr1 278 G ,,.,.,...,,,.,....^k. %38*<<;<7<<7<=<<<;<<<<<
chr1 279 C 23 A..T,,.,.,...,,,.,..... ;75&<<<<<<<<<=<<<9<<:<<

18. 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

19. UCSC Genome Browser with 1,000 Genomes Project Data

20. Integrated Genomics Viewer (IGV)

21. LookSeq at Sanger Mouse Genomes Project

22. Glo1 CNV Present in Mouse Genomes Data for A/J
Proximal Flank
Chr17: 30.5Mb
Max ~50x coverage
Glo1 Locus
Chr17: 30.7Mb
Max >100x coverage
Distal Flank
Chr17: 31.2Mb
Max ~50x coverage
50kb
50kb
50kb

23. Glo1 CNV Not Present in Mouse Genomes Data for NZO
Proximal Flank
Chr17: 30.5Mb
Max ~25x coverage
Glo1 Locus
Chr17: 30.7Mb
Max ~25x coverage
Distal Flank
Chr17: 31.2Mb
Max ~25x coverage
50kb
50kb
50kb

24. Galaxy (http://main.g2.bx.psu.edu)

25. Public Data Repositories
NCBI
EBI
SRA Formatted Files
FASTQ Files
Automatically Forward FASTQ Files to Galaxy
SRA ToolKit
FASTQ Files

26. NCBI BioProject

27. NCBI Gene Expression Omnibus

28. Overall Analysis Workflow
FASTQ Files
Secondary Analysis
Read Preprocessing & Diagnostics
Align Reads to Reference
Analysis of Aligned Reads
e.g., Read counts per gene from RNA-Seq
Tertiary Analysis
Analysis of Read Counts
e.g., Differentially expressed genes
Analysis of Gene Lists
Enrichment
Pathway and networks
Analysis of Expression Patterns

29. Push-Button Bioinformatics … Be Careful

30. Third Generation Sequencing