Getting nuclear genome

[bioman1]

I have plant genomic reads (illumina paired-end reads, Hiseq 2000 -WGS approach). I would like to get nuclear genome eliminating probable contaminating sequences (like mitochondrial, chloroplast, bacterial sequences and vector sequences). After eliminating, I would like to do denovo assembly.

I would like to know the good workflow for this. Please help me in suggesting good workflow. The planned workflow

1. I am planning to map the filtered illumina paired-ends reads (filtered through trimmomatic tool) to Arabidopsis thaliana mitochondira & choloroplast genome uisng BWA and filter unmapped reads using samtools.

2. The unmapped reads will be nuclear reads.Again vector and bacterial contamination is removed by mapping against univec database (http://www.ncbi.nlm.nih.gov/tools/vecscreen/univec/) and bacterial genomes (http://www.ncbi.nlm.nih.gov/genomes/...l_taxtree.html).

My question is , how can I map the reads

(i) To i need to index reference genome individually or can I combine (chloroplast and mitochondira genome as one reference)?. If I need to index separately, how can I get unmapped reads from choloroplast and mitochondria genome as paired-end fastq file?
(ii) After getting unmapped reads, how can I remove bacterial and vector contamination from the reads?

[luc]

Hi,

BBMap makes most of these steps a bit easier with dedicated scripts/tools.

http://seqanswers.com/forums/showthread.php?t=41057

There should be no problem concatenating mitochondrial and chloroplast sequences for these purposes. Which vectors would you expect other than the sequencing adapters?

[Jeremy]

It might be more effective to exclude mitochondrial sequence after the assembly, the mitochondria and chloroplast tend to form separate contigs anyway. Unless your plant species is Arabidopsis, only using the arabidopsis mitochondria and chloroplast genomes as a reference will likely miss a lot of mitochondrial sequence. Plant mitochondria can be very diverse in sequence content and size.