Page Comparison

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

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• Overviews
  
  • Wikipedia overview of NGS technologies
  • Broad Center GA Boot Camp
  • Early paper comparing NGS technologies (Liu et al., 2012)

• Technology intros

  • Illumina (Solexa) – most common "short" (< 300 bp) read sequencing
    

    • Short Illumina video

    • Longer Illumina video
    • 2016 Sequencing Technologies talk
  • Newer single molecule sequencing
    • Oxford Nanopore
    • PacBio SMRT system
      • PCR-free protocol
  • Single cell sequencing
    • 10x Genomics platform
  • Older technologies (less common now)
    

    • Life Technologies SOLiD (short reads in "colorspace")

    • Roche/454 – long (multi-Kb) reads often used in assemblies

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• Comparison of different aligners
  • by Heng Li, developer of bwa, samtools, and many other bioinformatics tools
• File formats
  • input: FASTQ format
  • output: the SAM (Sequence Alignment Map) format specification (SAM1.pdf)
• Aligners
  • bwa (Burrows-Wheeler Aligner) by Heng Li – http://bio-bwa.sourceforge.net/
    • fast, sensitive and easy to use
  • bowtie2 – http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml
    • fast, sensitive and extremely configurable
• The BioITeam has some TACC-aware alignment scripts you might find useful:
  • bwa alignment
    • /work/projects/BioITeam/common/script/align_bwa_illumina.sh
  • bowtie2 alignment
    • /work/projects/BioITeam/common/script/align_bowtie2_illumina.sh
  • merging sorted BAM files (read-group aware)
    
    • /work/projects/BioITeam/common/script/merge_sorted_bams.sh
  • email or come talk to Anna if you have questions or problems

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• SAM (Sequence Alignment Map) format specification (SAM1.pdf)
  
  • Translate SAM file flags web calculator: http://broadinstitute.github.io/picard/explain-flags.html
    
    • type in a decimal number to see which flags are set
• samtools – by Heng Li
  • SAM/BAM conversion, flag filtering, sorting, indexing, duplicate filtering
  • older 0.1.xx versions: http://samtools.sourceforge.net/
  • newer 1.3+ versions: http://www.htslib.org/
• Picard toolkit – http://broadinstitute.github.io/picard/
  • SAM/BAM utilities that are read-group aware
  • especially MarkDuplicates for flagging duplicate alignments
    • http://broadinstitute.github.io/picard/command-line-overview.html#MarkDuplicates
• bedtools – http://bedtools.readthedocs.org/en/latest/
  
  • All sub-commands: http://bedtools.readthedocs.io/en/latest/content/bedtools-suite.html
  • Swiss army knife for all manner of common BED, BAM, VCF, GFF/GTF file manipulation.
  • See BEDTools Overview for some common use cases.
  • Available in the TACC module system
• SAMStat - http://samstat.sourceforge.net/
  
  • produces detailed graphical statistics for SAM/BAM files.

File formats and conversion

• SAM format specification – http://samtools.github.io/hts-specs/SAMv1.pdf
  • crucial for performing format conversions, of which ChIP-seq analysis can have many
• Genome browser file formats – http://genome.ucsc.edu/FAQ/FAQformat.html
  
  • BED, bedGraph, narrowPeak and many more
• SRA (Sequence Read Archive) from NCBI
  • overview on this wiki
  • SRA search home page
  • SRA Toolkit
    • NCBI documentation
    • SRA toolkit downloads
• UCSC file format conversion scripts - useful for getting to/from WIG and BED to corresponding binary formats.
  • Make sure you download the correct scripts for your operating system!
  • Directories containing these tools can be found at TACC:
    • /work/projects/BioITeam/common/opt/UCSC_utils.2019_09
    • /work/projects/BioITeam/common/opt/UCSC_utils.2017_07
      

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RNAseq/Transcriptome analysis

• HISAT2, StringTie, BallGown suite – httpsGene quantification from BAM/BED file reads
  • featureCounts (part of the Subread package) – http://
  ccb
  • subread.
  jhu.edu/software/hisat2/index.shtml
  
  • from the Johns Hopkins group who brought you the Tuxedo pipeline – but much faster!
  • paper: http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.095.htmlsourceforge.net/
  • HTSeq – https://htseq.readthedocs.io/en/master/
• The Tuxedo pipeline: RNAseq with tophat/cufflinks
  • one of the first tool suites for transcriptome-aware RNA-seq alignment and quantification
  • RNAseq analysis protocol article in Nature Protocols
  • TopHat - http://ccb.jhu.edu/software/tophat/index.shtml
    
    • exon-aware sequence alignment (uses bowtie2/bowtie )
    • resource bundles for selected organisms (GFF annotations, pre-built bowtie2 references, etc.)
  • cuffquant, cuffnorm, cufflinks – http://cole-trapnell-lab.github.io/cufflinks/manual/
    • transcript quantification, normalization, differential expression
• HISAT2, StringTie, BallGown suite – https://ccb.jhu.edu/software/hisat2/index.shtml
  
  • from the Johns Hopkins group who brought you the Tuxedo pipeline – but much faster!
  • paper: http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.095.html
• General RNA-seq analysis workflow from Bioconductor:
• https://www.bioconductor.org/help/workflows/rnaseqGene/
• DESeq2 – R Bioconductor package
  
  • DESeq (version 1) documentation:
  • https://bioconductor.org/packages/release/bioc/vignettes/DESeq/inst/doc/DESeq.pdf
  • while DESeq2 is more sophisticated, reading the original documentation is a better introduction to concepts
  • DESeq2 documentation:
  • http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html
• kallisto – https://pachterlab.github.io/kallisto/
  • RNA-seq pseudoaligner that goes straight from FASTQ to estimated transcript abundances
    
    • blindingly fast – but only to transcriptome
  • companion quantification tool is sleuth – http://pachterlab.github.io/sleuth/about
  • overview presentation – 2015-10-21-Kallisto.Anna.pdf
• Dhivya Arasappan's Introduction to RNA Seq CCBB summer school course

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