Core NGS Tools -- Resource lists

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

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 bam files
2012 Next-Gen Sequence Analysis Workshop (Michigan State University) has similar tutorials to our course
- also includes introductions to using the Amazon EC2 where you can "rent" Linux machines (useful if you don't have access to TACC)
- Python, R, ChIP-Seq, etc.maintained mRNA-seq protocols from this workshop here

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://code.google.com/p/cutadapt/
- An excellent command line tool for adapter sequence removal
- Good support for trimming paired-end datasets
- Available at TACC only at Anna's /work/01063/abattenh/local/bin/cutadapt
- documentation at https://cutadapt.readthedocs.org/en/stable/
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

Comparison of different aligners
- by Heng Li, developer of BWA and MAQ
- by Nils Homer, developer of BFAST
File formats
- input: fastq format
- output: the SAM (Sequence Alignment Map) format specification (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
Anna has some TACC-aware alignment scripts you might find useful
- bwa alignment
  - /work/01063/abattenh/code/script/align/align_bwa_illumina.sh
- bowtie2 alignment
  - /work/01063/abattenh/code/script/align/align_bowtie2_illumina.sh
- merging sorted BAM files (read-group aware)
  - /work/01063/abattenh/code/script/align/merge_sorted_bams.sh
- email or come talk to me if you have questions or problems

SAM (Sequence Alignment Map) format specification (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 – http://samtools.sourceforge.net/ by Heng Li
- sam/bam conversion, flag filtering, sorting, indexing, duplicate filtering
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
SAMStat - http://samstat.sourceforge.net/
- produces detailed graphical statistics for sam/bam files.
bedtools – http://bedtools.readthedocs.org/en/latest/
- 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
  - merging overlapping regions
    - bedtools merge
  - generation of per-base genome-wide signal in bedGraph format
    - bedtools coverage
    - bedtools multicov
  - extracting fasta corresponding to regions
    - bedtools getfasta
- Available in the TACC module system

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!
- A directory containing these tools can be found on stampede at /work/01063/abattenh/local/UCSC_utilities
Mason program for simulating second-generation sequencing reads

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

The 1000 Genomes project - catalog of human genetic variants
Tools
- Broad institute GATK - complex but powerful; used by 1000 Genomes
File formats
- VCF (Variant Call Format) v4.0 - developed by 1000 Genomes project
  - list of tools and format specs