Page Comparison

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

Terminal programs

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• Command shell has ssh and scp (may require latest Windows updates)
  • Start menu → Search for Command
• Windows Subsystem for Linux – Windows 10 Professional includes a Ubuntu-like bash shells
  • See https://docs.microsoft.com/en-us/windows/wsl/install-win10
  • We recommend the Ubuntu Linux distribution, but any Linux distribution will have an SSH client

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• simple Terminal and file copy programs
• download either the Putty installer (https://the.earth.li/~sgtatham/putty/latest/w64/putty-64bit-0.70-installer.msi)
  • or just putty.exe (terminal) and pscp.exe (secure copy client)

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

Linux/TACC

• Linux fundamentals on this wiki
• Cloud recording of CBRS's Intro to Unix short course, taught by Benni Goetz:
  • https://utexas.zoom.us/rec/play/5co1OfvhN5650hOccTNzANod0hU7h-b8cFvCos8Y1Q_LTO3_PYfMfiQ6PjxaeYSJIwk4_8Sxag33qbMt.9qt5joWzzGbgRTSy?continueMode=true
• Cloud recording of CBRS's Intro to TACC short course, taught by Benni Goetz:
  • https://utexas.zoom.us/rec/share/x1zCXQg-nB1HZYzgnXb8VK44J0bSkxHQr1evth08tJEeqtfeGk366fVMtrV9NEVv.zoUqgtYhGTy2-FEv

• Online tutorials:

  • Ryan's Linux Tutorial: http://ryanstutorials.net/linuxtutorial/
  • Unix bootcamp for biologists: http://korflab.ucdavis.edu/bootcamp.html
  • Unix primer (longer version) for biologists:
    • http://korflab.ucdavis.edu/Unix_and_Perl/unix_and_perl_v2.3.4.pdf

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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 – header fields, body fields, flag definitions
    • https://github.com/samtools/hts-specs/blob/master/SAMtags.pdf – tag fields
• 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
    • /work2/projects/BioITeam/common/script/align_bwa_illumina.sh
  • bowtie2 alignment
    • /work2/projects/BioITeam/common/script/align_bowtie2_illumina.sh
  • merging sorted BAM files (read-group aware)
    
    • /work2/projects/BioITeam/common/script/merge_sorted_bams.sh
  • kallisto pseudo-alignment to annotated transcripts
    • /work2/projects/BioITeam/common/script/run_kallisto.sh
  • also available on many BRCF pods under /mnt/bioi/script.
  • 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
• RNA-seq QC, metrics & plotting tools:
  • RSeQC – http://rseqc.sourceforge.net/

  • RNA-SeQC (Broad Institute) –

    • https://software.broadinstitute.org/cancer/cga/rna-seqc
    • https://software.broadinstitute.org/cancer/cga/rnaseqc_run
  • RNA-QC-Chain – http://bioinfo.single-cell.cn/rna-qc-chain.html

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
• HTS format specifications – http://samtools.github.io/hts-specs/
  • clearinghouse page for a number of NGS formats (SAM, CRAM, VCF, BCF, etc.)
• Genome browser file formats – http://genome.ucsc.edu/FAQ/FAQformat.html
  
  • BED, bedGraph, narrowPeak and many more
• SRA (Sequence Read Archive) from NCBI
  • SRA search home page
  • SRA Toolkit
    • NCBI documentation
    • SRA toolkit downloads
• BioITeam script for converting GTF/GFF3 files to BED format
  • /work2/projects/BioITeam/common/script/gtf_to_bed.pl
• 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:
    • /work2/projects/BioITeam/common/opt/UCSC_utils.2019_09
    • /work2/projects/BioITeam/common/opt/UCSC_utils.2017_07
      

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