Page Comparison

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

...

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

• Technology intros

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

    • Official Short Illumina video

    • Another video (longer, good lecture)Longer Illumina video
    • 2016 Sequencing Technologies talk
  • Newer "single molecule" sequencing
    • PacBio SMRT system
      • PCR-free protocol
  • Older technologies (less common now)
    

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

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

...

• Comparison of different aligners
  • by Heng Li, developer of BWA and MAQ
• 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

...

• 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

...