Page Comparison

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

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• 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 (requires login after quick registration)
• Michigan State University ANGUS resources
  
  • A list of their tutorials: http://ged.msu.edu/angus/
  • 2012 Next-Gen Sequence Analysis Workshop
  (Michigan State University) has similar tutorials
  • a similar tutorial to our course
  • also includes introductions to using Introduction to Sequence analysis in the Amazon EC2 cloud
    
    • 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

Sequencing Technologies

• Paper comparing NGS technologies (Liu et al., 2012)

• Broad Center GA Boot Camp

• Technology videos

  • Illumina (Solexa)
    

    • Official video

    • Another video

  • Life Technologies SOLiD

  • Roche/454

  • Pacific Biosciences

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• Wikipedia FASTQ format page
• Illumina library construction on GSAF user wiki - useful for contaminant detection or adapter removal
• FastQC from Babraham Bioinformatics – http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
  
  • produces nice quality report for fastq files
• cutadapt – https://code.google.com/p/cutadapt/
  • An excellent command line tool for adapter sequence removal
  • Available at TACC only at Anna's /work/01063/abattenh/local/bin/cutadapt
  • documentation at https://cutadapt.readthedocs.org/en/stable/
  • Good support for trimming paired-end datasets
  • Anna has an script that handles the details of paired-end read trimming:
    • /work/01063/abattenh/code/script/align/trim_adapters.sh
• trimmomatic – http://www.usadellab.org/cms/?page=trimmomatic
  • Supports trimming paired-end datasets. I haven't used it but it seems to be popular.
• fastx toolkit – http://hannonlab.cshl.edu/fastx_toolkit/
  • Command line tools for fastq analysis and manipulation
  • Good for hard clipping. Available at TACC.
  • Documentation at: http://hannonlab.cshl.edu/fastx_toolkit/commandline.html

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• Comparison of different aligners
  • by Heng Li, developer of BWA and MAQ
  • by Nils Homer, developer of BFAST
• File formats
  • input: fastq format
  • output: the SAM (Sequence Alignment Map) format specification (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
• Anna has some TACC-aware alignment scripts you might find useful
  • bwa alignment
    • /work/01063/abattenh/code/script/align/align_bwa_illumina.sh
  • bowtie2 alignment
    • /work/01063/abattenh/code/script/align/align_bowtie2_illumina.sh
  • merging sorted BAM files (read-group aware)
    
    • /work/01063/abattenh/code/script/align/merge_sorted_bams.sh
  • email or come talk to me if you have questions or problems

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• SAM (Sequence Alignment Map) format specification (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 – http://samtools.sourceforge.net/ by Heng Li
  
  • sam/bam conversion, flag filtering, sorting, indexing, duplicate filtering
• 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
• SAMStat - http://samstat.sourceforge.net/
  
  • produces detailed graphical statistics for sam/bam files.
• bedtools – http://bedtools.readthedocs.org/en/latest/
  
  • Swiss army knife for all manner of common bed, bam, vcf, gff file manipulation such as:
    • intersecting bam or bed with annotation files
      • bedtools intersect
    • merging overlapping regions
      • bedtools merge
    • generation of per-base genome-wide signal in bedGraph format
      • bedtools coverage 
      • bedtools multicov
    • extracting fasta corresponding to regions
      • bedtools getfasta
  • Available in the TACC module system

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 script for your operating system!
  • 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

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