Content Comparison

A healthy taste of resources available, specifically for this course - not a comprehensive catalog.

Sequencing Technologies

• Paper comparing NGS technologies (Liu et al., 2012)
• Broad Center GA Boot Camp

• Technology videos

  • Roche/454

  • Illumina (Solexa) Genome Analyzer and HiSeq

  • Life Technologies SOLiD

  • Pacific Biosciences

Community Resources

• SEQAnwers forum - many NGS sequencing questions answered here
  • A funny SEQAnwers post about biologists starting to analyze NGS data: http://seqanswers.com/forums/showthread.php?t=4589
• UCSC Genome Browser - visualize and download NGS data (see more below)
• Galaxy website for online sequencing data analysis
• Broad Institute Integrated Genomcs Viewer (IGV) - especially good for bam files

Getting started with Linux and NGS

• Cheat sheet of useful Unix commands
• A funny SEQAnwers post about biologists starting to analyze NGS datahttp://seqanswers.com/forums/showthread.php?t=45892012 Next-Gen Sequence Analysis Workshop (Michigan State University) has similar tutorials to our course
  • also includes introductions to using the Amazon EC2 where you can "rent" Linux machines (useful if you don't have access to TACC)
  • Python, R, ChIP-Seq, etc.maintained mRNA-seq protocols from this workshop here

Fastq analysis/manipulation

• Wikipedia FASTQ format page
• FastQC from Babraham Bioinformatics; produces nice quality report for fastq files.
• Cutadapt - An excellent command line tool for adapter sequence removal.
• FASTX Toolkit - Command line tools for fastq analysis and manipulation
• Illumina library construction on GSAF user wiki - useful for contaminant detection or adapter removal.

Alignment and aligners

• Jeff Barrick's introduction to NGS presentation
  • 
• Comparison of different aligners
  • by Heng Li, developer of BWA and MAQ
  • by Nils Homer, developer of BFAST
• Aligners
  • bowtie (http://bowtie-bio.sourceforge.net/index.shtml) - very fast, not very sensitive
  • BFAST wiki & manual - slow and relatively complicated, but tunable sensitivity
  • bwa - fast, sensitive and easy to use
  • bowtie2 - fast, sensitive, configurable, easy to use
• File formats
  • fastq format
  • The SAM (Sequence Alignment Map) format specification (pdf)

Alignment analysis

• SAM (Sequence Alignment Map) format specification (pdf)
• sam/bam tools
  • samtools - sam/bam conversion, flag filtering, bam sort/index
  • Picard - sam/bam utilities that are read-group aware
  • Translate SAM file flags - type in a decimal number to see which flags are set
• SAMstat - produces detailed graphical statistics for sam/bam files.
• BEDTools - region overlap, merge, coverage & much more, w/bed, bam, vcf, gff support
  • BEDTools manual on readthedocs.org

UCSC Genome Browser

• intro on this wiki
• Main UCSC Genome Browser web site
• Beta Test browser site - most up-to-date datasets and features; can be buggy
• File formats - BED format especially is widely used
• Table browser - Browse and download data in different formats
• ENCODE data downloads at UCSC - useful for getting data to work with

Variant calling

• The 1000 Genomes project - catalog of human genetic variants
• Tools
• Broad institute GATK - complex but powerful; used by 1000 Genomes
File formatsVCF (Variant Call

Format

...

• list of tools and format specs

Transcriptome analysis

• The Tuxedo pipeline: RNAseq with tophat/cufflinks
  • tophat - exon-aware sequence alignment (uses bowtie)
  • cufflinks - transcript assembly, differential expression & regulation
  • RNAseq analysis protocol article in Nature Protocols
    • cufflinks resource bundles for selected organisms (gff annotations, pre-built bowtie references, etc.)

...

converters and generation tools

• SRA (Sequence Read Archive) from NCBI
  • overview on this wiki
  • SRA search home page
  • SRA Toolkit
    • NCBI documentation
    • SRA toolkit downloads
  Mason program for simulating second-generation sequencing reads.
• UCSC file format conversion scripts - useful for getting to/from wig and bed to corresponding binary formats.
  • Make sure you download the correct script for your operating system!

Other courses with online tutorials

• 2012 Next-Gen Sequence Analysis Workshop (Michigan State University) has similar tutorials to our course, but also includes introductions to using the Amazon EC2 where you can "rent" Linux machines (useful if you don't have access to TACC), Python, R, ChIP-Seq, etc.
  • maintained mRNA-seq protocols from this workshop here
  • A directory containing these tools can be found on stampede at /work/01063/abattenh/local/UCSC_utilities
• Mason program for simulating second-generation sequencing reads.

Transcriptome analysis

• The Tuxedo pipeline: RNAseq with tophat/cufflinks
  • RNAseq analysis protocol article in Nature Protocols
  • tophat - exon-aware sequence alignment (uses bowtie2/bowtie )
  • cufflinks - transcript assembly, differential expression & regulation
    • cufflinks resource bundles for selected organisms (gff annotations, pre-built bowtie2 references, etc.)

Variant calling

• The 1000 Genomes project - catalog of human genetic variants
• Tools
  • Broad institute GATK - complex but powerful; used by 1000 Genomes
• File formats
  • VCF (Variant Call Format) v4.0 - developed by 1000 Genomes project
    • list of tools and format specs