2020 Core NGS Resources

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

Linux

• 2020 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

• 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

  • 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

FASTQ analysis/manipulation/QC

• 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

• MultiQC – http://multiqc.info/

  • A great tool for consolidating QC multiple QC reports into one HTML page

  • Anna's Byte Club tutorial on using MultiQC – https://utexas.atlassian.net/wiki/display/bioiteam/Using+MultiQC

  • Available on ls5 at /work/projects/BioITeam/ls5/opt/multiqc-1.0/multiqc

    • also needs this $PYTHONPATH modification:
      export PYTHONPATH="/work/projects/BioITeam/ls5/lib/python2.7/annab-packages:$PYTHONPATH"

• cutadapt – https://cutadapt.readthedocs.io/en/stable/

  • An excellent command line tool for adapter sequence removal

  • Good support for trimming paired-end datasets

  • Available on ls5 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.

• fastx toolkit – http://hannonlab.cshl.edu/fastx_toolkit/

  • Suite of command line tools for FASTQ and FASTA analysis and manipulation

  • Good for hard clipping. Available at TACC.

  • Documentation at: http://hannonlab.cshl.edu/fastx_toolkit/commandline.html

• seqtk – https://github.com/lh3/seqtk

  • Suite of command line tools for FASTQ and FASTA analysis and manipulation

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

  • RefSeq – https://www.ncbi.nlm.nih.gov/refseq/

    • well curated genome, transcriptome sequences

  • GenBank – https://www.ncbi.nlm.nih.gov/genbank/

    • public repository for sequence data, especially for prokaryotic genomes

    • not curated

• 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 bioinformatics tools

• File formats

  • input: FASTQ format

  • output: the SAM (Sequence Alignment Map) format specification ()

• 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

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 RNA-seq 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 ()

  • 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

• 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.2019_09

    • /work/projects/BioITeam/common/opt/UCSC_utils.2017_07

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

• 2020 Visualize mapped data at UCSC genome browser on this wiki

RNAseq/Transcriptome analysis

• Gene quantification from BAM/BED file reads

  • featureCounts (part of the Subread package) – http://subread.sourceforge.net/

  • HTSeq – https://htseq.readthedocs.io/en/master/

• The Tuxedo pipeline: RNAseq with tophat/cufflinks

  • one of the first tool suites for transcriptome-aware RNA-seq alignment and quantification

  • 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

• 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

• 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 – https://pachterlab.github.io/kallisto/

  • RNA-seq pseudoaligner that goes straight from FASTQ to estimated transcript abundances

    • 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

• 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/splash.php

  • Genomic Regions Enrichment of Annotations Tool