Core NGS Resources

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

Linux

• Linux fundamentals on this wiki

• Online tutorials:

  • Ryan's Linux Tutorial: http://ryanstutorials.net/linuxtutorial/
  • Unix and Perl for Biologists: http://korflab.ucdavis.edu/unix_and_Perl/

Community Resources

• 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 visualizing bam file details
• Michigan State University ANGUS resources
  
  • A list of their tutorials: http://ged.msu.edu/angus/
  • 2012 Next-Gen Sequence Analysis Workshop a similar tutorial to our course
  • Introduction to Sequence analysis in the Amazon EC2 cloud
    
    • where you can "rent" Linux machines (useful if you don't have access to TACC)

Sequencing Technologies

• 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

Fastq analysis/manipulation

• 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://cutadapt.readthedocs.io/en/stable/
  • An excellent command line tool for adapter sequence removal
  • Good support for trimming paired-end datasets
  • Available at TACC at /work/projects/BioITeam/ls5/opt/cutadapt-1.10/cutadapt
    • also needs this $PYTHONPATH modification:
      export PYTHONPATH="/work/projects/BioITeam/ls5/lib/python2.7/site-packages:$PYTHONPATH"
  • Script that handles the details of paired-end read trimming
    
    • /work/projects/BioITeam/common/script/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

Reference genomes

• Gencode – https://www.gencodegenes.org/
  
  • reference genomes, transcriptomes and high-quality annotations for human and mouse
  • https://www.gencodegenes.org/releases/current.html
• UCSC downloads – http://hgdownload.cse.ucsc.edu/downloads.html
  • reference genomes, transcriptomes and high-quality annotations for many eukaryotes
• Ensembl downloads – ftp://ftp.ensembl.org/pub/
  • reference genomes, transcriptomes and high-quality annotations for many eukaryotes
• NCBI GenBank Nucleotide collection – https://www.ncbi.nlm.nih.gov/nuccore/
  • for prokaryotic genomes
• Reference genome vocabulary – https://software.broadinstitute.org/gatk/documentation/article?id=7857
  • excellent introduction to the types of genome references and the vocabulary used to describe them
    • aimed at higher eukaryotes but vocabulary useful nonethele
• GATK blog describing ALT contigs in GRCh38 – https://software.broadinstitute.org/gatk/blog?id=8180
• Support for mapping to ALT contigs containing variants
  
  • bwa mem + bwakit by Heng-Li – https://github.com/lh3/bwa/blob/master/README-alt.md

Basic alignment and aligners

• Comparison of different aligners
  • by Heng Li, developer of BWA, samtools, and many other
• 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 me if you have questions or problems

Transcriptome-aware aligners

• HISAT2 – https://ccb.jhu.edu/software/hisat2/index.shtml
  
  • new and fast, with support for alignment to single and "population" of genomes
  • paper: http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.095.html
• STAR (Spliced Transcripts Alignment to a Reference) – ultra-fast RNAseq aligner
  • releases: https://github.com/alexdobin/STAR/releases
  • paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530905/
  • STAR manual (pdf)
• TopHat - http://ccb.jhu.edu/software/tophat/index.shtml
  
  • exon-aware sequence alignment (uses bowtie2/bowtie )
• kallisto - https://pachterlab.github.io/kallisto/about
  • ultra-fast RNA-seq pseudoaligner that goes straight from fastq to estimated transcript abundances

Alignment analysis

• 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
    
  • 0.1.xx versions: http://samtools.sourceforge.net/
  • 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 file manipulation such as:
    • intersecting bam or bed with annotation files
      • bedtools 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 format
      • bedtools 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 regions
      • bedtools 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)
        
  • 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!
  • Directories containing these tools can be found on ls5 at
    •  /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

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

RNAseq/Transcriptome analysis

• HISAT2, StringTie, BallGown suite – https://ccb.jhu.edu/software/hisat2/index.shtml
  
  • from the Johns Hopkins group who brought you the Tuxedo pipeline – but much faster!
  • paper: http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.095.html
• The Tuxedo pipeline: RNAseq with tophat/cufflinks
  • RNAseq analysis protocol article in Nature Protocols
  • TopHat - http://ccb.jhu.edu/software/tophat/index.shtml
    
    • exon-aware sequence alignment (uses bowtie2/bowtie )
    • resource bundles for selected organisms (gff annotations, pre-built bowtie2 references, etc.)
  • cuffquant, cuffnorm, cufflinks – http://cole-trapnell-lab.github.io/cufflinks/manual/
    • transcript quantification, normalization, differential expression
• General RNA-seq analysis workflow from Bioconductor:
• https://www.bioconductor.org/help/workflows/rnaseqGene/
• DESeq2 – R Bioconductor package
  
  • DESeq (version 1) documentation:
  • https://bioconductor.org/packages/release/bioc/vignettes/DESeq/inst/doc/DESeq.pdf
  • while DESeq2 is more sophisticated, reading the original documentation is a better introduction to concepts
  • DESeq2 documentation:
  • http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html
• kallisto pseudo-alignment – https://pachterlab.github.io/kallisto/
  • blindingly fast – but only to transcriptome
  • companion quantification tool is sleuth – http://pachterlab.github.io/sleuth/about
  • overview presentation – 2015-10-21-Kallisto.Anna.pdf
• Dhivya Arasappan's Introduction to RNA Seq CCBB summer school course

Variant calling

• Tools
  • Broad institute GATK - complex but powerful; used by TCGA, 1000 Genomes
    • documentation page: https://software.broadinstitute.org/gatk/documentation/
• 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

• DAVID – functional annotation from user-supplied gene lists
• GREAT – analysis tool that takes bed files as input and outputs enriched genes, GO-terms, motifs, etc.
  • for human, mouse, zebrafish
• MEME-suite – a motif identification and discovery tool.  Works with most species.
  • takes fasta files as input, so filter your bam/bed files to get the regions of interest, then convert over using bamtofastq in bedtools.