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
  • 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 (mult-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.x+ versions: http://www.htslib.org/
• Picard toolkit – http://broadinstitute.github.io/picard/
  • SAM/BAM utilities that are read-group aware
  • especially MarkDuplicates and MarkDuplicatesWithMateCigar for flagging duplicate alignments
    • http://broadinstitute.github.io/picard/command-line-overview.html#MarkDuplicates
    • http://broadinstitute.github.io/picard/command-line-overview.html#MarkDuplicatesWithMateCigar
• SAMStat - http://samstat.sourceforge.net/
  
  • produces detailed graphical statistics for SAM/BAM files.
• 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 such as:intersecting BAM or BED with annotation filesbedtools intersect (http://bedtools.readthedocs.io/en/latest/content/tools/intersect.html)
    
  • merging overlapping regions
    • bedtools merge (http://bedtools.readthedocs.io/en/latest/content/tools/merge.html)
      
  generation of per-base genome-wide signal in bedGraph formatbedtools coverage(http://bedtools.readthedocs.io/en/latest/content/tools/coverage.html)bedtools multicov(http://bedtools.readthedocs.io/en/latest/content/tools/multicov.html)extracting FASTA corresponding to regionsbedtools getfasta (http://bedtools.readthedocs.io/en/latest/content/tools/getfasta.html) format conversion
  • bedtools bamtobed (http://bedtools.readthedocs.io/en/latest/content/tools/bamtobed.html)
  • bedtools bamtofastq (http://bedtools.readthedocs.io/en/latest/content/tools/bamtofastq.html)
    
  bedtools bedtobam (http://bedtools.readthedocs.io/en/latest/content/tools/bedtobam.html)
  • See BEDTools Overview for some common use cases.
  • 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 scripts for your operating system!
  • Directories containing these tools can be found at TACC:
    •  /work/projects/BioITeam/common/opt/UCSC_utils.2013_03
    • /work/projects/BioITeam/common/opt/UCSC_utils.2017_07
      
• Mason program for simulating NGS sequencing reads

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