Page History

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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://wikis.utexas.edu/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.
- 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

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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
  - /work2work/projects/BioITeam/common/script/align_bwa_illumina.sh
- bowtie2 alignment
  - /work2work/projects/BioITeam/common/script/align_bowtie2_illumina.sh
- merging sorted BAM files (read-group aware)
  - /work2work/projects/BioITeam/common/script/merge_sorted_bams.sh
- kallisto pseudo-alignment to annotated transcripts
  - /work2work/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

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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
- Also available as a BioContainers module

UCSC 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

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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 Differential Gene Expression (DEG) analysis workflow from R's Bioconductor:

DESeq2 – R Bioconductor package
- DESeq (version 1) documentation:
- DESeq2 documentation:
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 CBRS 2021 summer school course

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 CBRS 2021 summer school course

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