Core NGS Resources

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

Linux/TACC

Linux fundamentals on this wiki
Wikis for the 3 CBRS Unix/Linux workshops:
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

Community Resources

UCSC Genome Browser - visualize and download NGS data (see more below)
Broad Institute Integrated Genomics 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 or BRCF pods)
Galaxy website for online sequencing data analysis
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

Sequencing Technologies

Overviews
Technology intros
- Illumina (Solexa) – most common "short" (< 300 bp) read sequencing
- Newer single molecule sequencing
  - Oxford Nanopore
  - PacBio SMRT system
- Single cell sequencing
  - 10x Genomics platforms
- Older technologies (not 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
cutadapt – https://cutadapt.readthedocs.io/en/stable/
- An excellent command line tool for adapter sequence removal
- Good support for trimming paired-end datasets
- Script that handles the details of paired-end read trimming
  - /work2/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, FASTA file manipulations
- 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
UCSC downloads – http://hgdownload.cse.ucsc.edu/downloads.html
- reference genomes, transcriptomes and high-quality annotations for many eukaryotes
Ensembl downloads – http://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 nonetheless
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

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
Comparison of different aligners
- by Heng Li, developer of bwa, samtools, and many other bioinformatics tools
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
- kallisto pseudo-alignment to annotated transcripts
  - /work/projects/BioITeam/common/script/run_kallisto.sh
- also available on many BRCF pods under /mnt/bioi/script.
  - many pre-built references also available in /mnt/bioi/ref_genome
- email or come talk to Anna if you have questions or problems

Transcriptome-aware aligners

HISAT2 – https://daehwankimlab.github.io/hisat2/
- 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: https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.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
- 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
- /work/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!
- Also available as a BioContainers module

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

RNAseq/Transcriptome analysis

General RNA-seq Differential Gene Expression (DGE) analysis workflow from R's Bioconductor:
- https://www.bioconductor.org/help/workflows/rnaseqGene/
Gene quantification from BAM/BED file reads
- featureCounts (part of the Subread package) – http://subread.sourceforge.net/
- HTSeq – https://htseq.readthedocs.io/en/master/
HISAT2, StringTie, BallGown suite – https://ccb.jhu.edu/software/hisat2/index.shtml
- transcriptome-aware alignment & quantification 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
DESeq2 – R Bioconductor package for DGE
- 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
The Tuxedo pipeline: RNAseq with tophat/cufflinks
- one of the first tool suites for transcriptome-aware RNA-seq alignment and quantification
  - rarely used now, as other tools are much faster & more accurate
- 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
Dhivya Arasappan's Introduction to RNA Seq CBRS 2021 summer school course

Variant calling

Broad institute GATK (Genome Analysis Tool Kit) – https://software.broadinstitute.org/gatk/documentation/
- complex but powerful
- used by TCGA (The Cancer Genome Atlas), 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 CBRS 2021 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
GSEA – https://www.gsea-msigdb.org
- Gene Set Enrichment Analysis
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
- 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.