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.x3+ versions: http://www.htslib.org/
• Picard toolkit – http://broadinstitute.github.io/picard/
  • SAM/BAM utilities that are read-group aware
  • especially MarkDuplicatesand 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.
  • 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.20132019_0309
    • /work/projects/BioITeam/common/opt/UCSC_utils.2017_07
      
• Mason program for simulating NGS sequencing reads

UCSC Genome Browser

• Main UCSC Genome Browser web site
  • 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
  • Beta Test browser site - most up-to-date datasets and features; can be buggy
• Visualize mapped data at UCSC genome browser on this wiki

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• Broad institute GATK (Genome Analysis Tool Kit) – https://software.broadinstitute.org/gatk/documentation/
  
  • complex but powerful
  • used by TCGA, 1000 Genomes
• File formats
  • VCF (Variant Call Format) v4.0 - initially developed by 1000 Genomes project
  • MAF (Mutation Annotation Format) – developed by The Cancer Genome Atlas (TCGA)
• The International Genome Sample Resource – follow-on to the 1000 Genomes project 
  
  • catalog of human genetic variants
• Dan Deatherage's Genome Variant Analysis CCBB summer school course

Genome Annotation

• GO – http://geneontology.org/
  • The Gene Ontology resource, a large source of information on the functions of genes
• GOrilla – http://cbl-gorilla.cs.technion.ac.il/
  • Gene Ontology enRIchment anaLysis and visuaLizAtion tool
• DAVID – https://david.ncifcrf.gov/
  
  • functional annotation from user-supplied gene lists
• GREAT – http://bejerano.stanford.edu/great/public/html/
  analysis tool that splash.php
  
  • Genomic Regions Enrichment of Annotations Tool
  • takes bed files as input and outputs enriched genes, GO-terms, motifs, etc.
    • human, mouse, zebrafish
• MEME-suite – http://meme-suite.org/
  
  • a motif identification and discovery tool.  Works with most species.
  • takes FASTA files as input
    • filter your BAM/BED files to get the regions of interest
    • then convert to FASTA using bedtools bamtofastq.

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