Blaxter Lab, Institute of Evolutionary Biology, University of Edinburgh

Goal: To create blobplots or Taxon-Annotated-GC-Coverage plots (TAGC plots) to visualise the contents of genome assembly data sets as a QC step.

This repository accompanies the paper:
Blobology: exploring raw genome data for contaminants, symbionts and parasites using taxon-annotated GC-coverage plots. Sujai Kumar, Martin Jones, Georgios Koutsovoulos, Michael Clarke, Mark Blaxter
(submitted 2013-10-01 to Frontiers in Bioinformatics and Computational Biology special issue : Quality assessment and control of high-throughput sequencing data).

It contains bash/perl/R scripts for running the analysis presented in the paper to create a preliminary assembly, and to create and collate GC content, read coverage and taxon annotation for the preliminary assembly, which can be visualised, such as Figure 2a from the paper showing TAGC plots/blobplots for Caenorhabditis sp. 5: 

Address of the bookmark: https://github.com/blaxterlab/blobology

The pipeline consists of three steps/modules:

• redundancy reduction: detection and selectively removal of redundant contigs from an initial de novo assembly
• scaffolding: joining of genome fragments using paired-end and/or mate-pairs reads
• gap closing

Redundans is:

• fast & lightweight, multi-core support and memory-optimised, so it can be run even on the laptop for small-to-medium size genomes
• flexible toward many sequencing technologies (Illumina, 454 or Sanger) and library types (paired-end, mate pairs, fosmids)
• modular: every step can be ommited or replaced by another tools

Address of the bookmark: https://github.com/Gabaldonlab/redundans

https://github.com/aalokaily/Hierarchical-Genome-Assembly-HGA

Address of the bookmark: https://github.com/aalokaily/Hierarchical-Genome-Assembly-HGA

• velvet (velveth velvetg should be in your PATH)
• R (with Sweave)
• pdflatex (usually part of TeTeX)
• ggplot2 (from R prompt type install.packages("ggplot2","proto","xtable"))
• Perl

Optional:

• BLAT or BLAST (to generate alignments against a reference genome). If using BLAT, add faToTwoBit,gfClient,gfServer to your PATH. If using BLAST, add blastall and formatdb.

Edit permute.sh to your liking, paying particular attention to the kmer, cvCut, expCov, and other flags

To Run:

1. perl fastaAllSize mysequences.fa > mysequences.stat or gunzip -c mysequences.fa.gz | fastaAllSize > mysequences.stat Substitute fastqAllSize for fastq files.
2. ./permute.sh mysequences (leave out the .fa)

https://github.com/leipzig/standardized-velvet-assembly-report

Address of the bookmark: https://github.com/leipzig/standardized-velvet-assembly-report

The arguments accepted by ScaffMatch are:

  -w) Working directory -- this is the directory where ScaffMatch files are stored. These are .sam files produced after mapping reads to contigs and the resulting scaffolds file `scaffolds.fa` fasta file;

  -c) Contig fasta file;

  -m) Command line argument with no options. It is used when .sam files are used instead of reads .fastq files. Do not use this option if you provide reads files;

  -1) (Comma separated list of) either .fastq or .sam file(s) corresponding to the first read of the read pair;

  -2) (Comma separated list of) either .fastq or .sam file(s) corresponding to the second read of the read pair;

  -i) (Comma separated list of) insert size(s) of the library(-ies);

  -s) (Comma separated list of) library(-ies) standard deviation(s) of insert size(s);

  -t) Bundle threshold. Pairs of contigs supported by number of read pairs less than the value of this argument are discarded. Optional argument, by default it is equal to 5;

  -g) Matching heuristics: use `max_weight` for Maximum Weight Matching heuristics with the Insertion step, use `backbone` for Maximum Weight Matching heuristics without the Insertion step, use `greedy` for Greedy Matching heuristics;

  -l) Log file - where to store the logs. Optional argument. By default, stdout is used.

Address of the bookmark: http://alan.cs.gsu.edu/NGS/?q=content/scaffmatch

PEAR evaluates all possible paired-end read overlaps and without requiring the target fragment size as input. In addition, it implements a statistical test for minimizing false-positive results. Together with a highly optimized implementation, it can merge millions of paired end reads within a couple of minutes on a standard desktop computer.

Address of the bookmark: http://sco.h-its.org/exelixis/web/software/pear/doc.html

More at http://www.homolog.us/Tutorials/index.php?p=1.4&s=1

Address of the bookmark: http://www.homolog.us/Tutorials/index.php?p=1.4&s=1

Tools

• Parsnp - Core-genome alignment and analysis
• Gingr - Interactive visualization of alignments, trees and variants
• HarvestTools - Archiving and postprocessing

Citation

Treangen TJ, Ondov BD, Koren S, Phillippy AM. The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes. Genome Biology, 15 (11), 1-15 [PDF]

Address of the bookmark: http://harvest.readthedocs.io/en/latest/index.html

bedtools is developed in the Quinlan laboratory at the University of Utah and benefits from fantastic contributions made by scientists worldwide.

Address of the bookmark: http://bedtools.readthedocs.io/en/latest/index.html

Using the reference sequence dictionary (*.dict), it also creates some empty BAM files if no sam record was found for a chromosome. A pair of 'mock' SAM-Records can also be added to those empty BAMs to avoid some tools (like samtools) to crash.

Usage

java -jar splitbam.jar -p OUT/__CHROM__/__CHROM__.bam -R ref.fasta (bam|sam|stdin)

Options

• -h help; This screen.
• -R (indexed reference file) REQUIRED.
• -u (unmapped chromosome name): default:Unmapped
• -e | --empty : generate EMPTY bams for chromosome having no read mapped
• -m | --mock : if option '-e', add a mock pair of sam records to the empty bam
• -p (output file/bam pattern) REQUIRED. MUST contain __CHROM__ and end with .bam
• -s assume input is sorted.
• -x | --index create index.
• -t | --tmp (dir) tmp file directory
• -G (file) chrom-group file (see below)

Address of the bookmark: https://code.google.com/archive/p/jvarkit/wikis/SplitBam.wiki