RRedon:Protocols/Variation pipeline

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Get Reference Genome

Download the hg18/build36 from UCSC: http://hgdownload.cse.ucsc.edu/goldenPath/hg18/chromosomes

get SamTools

http://samtools.sourceforge.net/

With BWA

• http://bio-bwa.sourceforge.net

Merge all the reference sequences into one fasta file hg18.fasta (? ← Fix this! need merge ?) Index the reference genome:

  bwa index -a bwtsw hg18.fasta 

Pre mapping

← Fix this! why do we need a pre-mapping ?

• extract every 500th read from fastq file
• Align one fastq files

-l Take the first INT subsequence as seed

-q Parameter for read trimming.

 bwa aln -l 32 -q 15 -foutput1.aln hg18.fasta file1.fastq.gz
 bwa aln -l 32 -q 15 -foutput2.aln hg18.fasta file2.fastq.gz

• Generate alignments in the SAM format given paired-end reads. Repetitive read pairs will be placed randomly.

 bwa sampe hg18.fasta output1.aln output2.aln file1.fastq.gz file2.fastq.gz >  output.sam 

• export to bam

 samtools view output.sam >  output.bam 

• sort bam

 samtools sort output.bam sorted_prefix 

do insert size stats e.g. 99.8 percentile for MAQ max insert size ← Fix this! what does that mean ?

With MAQ

• http://maq.sourceforge.net/