Split fastq mates

Here is code in perl to split a single paired interleaved FASTQ file into a pair of FASTQ files:

#!/usr/local/bin/perl -w
# Daniel Brami
# Util to split interlaced FASTQ files into pairs

use strict;

# Standard lib
use IO::File;
use File::Basename;

my $INPUT=shift;

if (!(defined ($INPUT)) || ($INPUT =~ '^\-')){
die "Usage: $0 <interleaved paired FASTQ file>\n";
}

my ($name,$path,$suffix) = fileparse($INPUT, qw/fastq FASTQ txt TXT/);

my $FH_IN = new IO::File($INPUT, "r") or die "could not open $INPUT: $!\n";
my $FH_OUT1 = new IO::File($name."split1.$suffix", "w") or die "could not open $name.split1$suffix for writing: $!\n";
my $FH_OUT2 = new IO::File($name."split2.$suffix", "w") or die "could not open $name.split2$suffix for writing: $!\n";

my ($recs1, $recs2)= (0,0);
my $flipflop = 1;
my $counter = 0;
my ($line, $TXT);
while($line = $FH_IN->getline()){
$TXT .= $line;
$counter++;
if($counter == 4){
 if($flipflop == 1){
  print $FH_OUT1 $TXT;
  ++$recs1;
 }else{
  print $FH_OUT2 $TXT;     
  ++$recs2;
 }
 $counter = 0;
 $TXT = '';
 $flipflop *= -1;
}
}
$FH_IN->close();
$FH_OUT1->close();
$FH_OUT2->close();

print STDERR "Processed $recs1 records for pair file 1 and $recs2 records for pair file 2.\n";
if($recs1 != $recs2){
print STDERR "The number of processed records does not match - check input data!";
}exit;

Quality trimming fastq files using CLC Bio cell

1. copy the mandatory license.properties file to your working directory
2. /usr/local/packages/clc-ngs-cell/quality_trim -o -p -c -l -m -r -i &
3. Optional: Split the resulting interleaved paired file into two components:
   ~/workspace/meta_scripts/splitMatedFastq.pl

modify a genbank file for mapping

Insert between the following tags at the tail of your Genbank file the nucleotide FASTA sequence only:

ORIGIN

(sequence goes here)

//

CLC mapping

At ths point, the most current version of the CLC command-line utils is located here:

/usr/local/packages/clc-ngs-cell/

with the mapper for 454 / Sanger reads being clc_ref_assemble_long.

As usual, you need to copy the license.properties file from that folder to your working directory to perform any sort of analysis.

Here are some of the pitfalls:

- making a soft link to the mapping executable will throw a missing license error. forget the soft link, you need to use the fully qualified path of the executable.

- mapping to a Genbank file didn't work. However mapping to a FASTA file completed with blazing quickness.

Once the mapping has producea CAS output, you need to process it to get it in table format:

/usr/local/packages/clc-ngs-cell/assembly_table -n -s input.cas > output.table

SVN stuff

This is how you create a repository (usually done by IT):

svnadmin create (repo_name)

This is how you add content to a repository:

svn import (files | folder) (repo_name) -m "initial import"

This is how you checkout the latest version of something existing in SVN into the current working directory:

svn checkout (repo_name/path)

This is how you check something into it to update the svn with a more current version:

from within the root folder when doing svn checkout (ie: /workspace/genomeQC):

svn commit -m "update to ver 1.5"

FASTQ tools

Here are my favorite FASTQ processing utils:

FASTQC:

To run a QC on your data.

http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/

To run in batch mode with zipped output:

java* -Xmx250m -cp <> uk.ac.bbsrc.babraham.FastQC.FastQCApplication <>

*: make sure that java is at least version 1.6

FASTX Toolkit:

Bunch of scripts for processing the fastq files and qc, but fastqc is better for qc'ing.

http://hannonlab.cshl.edu/fastx_toolkit/download.html

Illumina Technote: How Illumina Sequencing works

Here's a a PDF that describes how Illumina seqeuncing works with nice graphics:
http://www.illumina.com/documents/products/datasheets/datasheet_genomic_sequence.pdf

A copy is saved here:

http://sites.google.com/site/webhostingoffiles/Illumina_datasheet_genomic_sequence.pdf

Another great article detailing sequencing technology is saved here:

http://sites.google.com/site/webhostingoffiles/Sequencingtechnologies%E2%80%94NextGen_nrg2626.pdf