How analyze DNA's reads by HiSeq-2000?

[vitor]

I need to analyze reads of viral DNA by deep sequencing using HiSeq-2000. I'll check the substitutions throughout the genome and identify the viral virants, but i don't know what package or scripts to use. Do you have any suggestions, friends?

Thanks.
Vitor

[jwfoley]

What kind of data do you have so far ("raw" FASTQ or is it already processed?) and what exactly do you want to achieve?

[vitor]

jwfoley, the data's still being processed and I'll receive in FASTQ. I want to find mutation hot spots, nucleotide substituations and comparing the diversity of the genome at various times.

[diego diaz]

Hi vitor,

First, you need to trim your reads, to discard low quality bases. You could use Trimmomatic, fastx_toolkit, prinseq, etc. Then, you have to map your sequences to the reference genome. If you want to analyze substitutions, I think it is more suitable to chose a more sensitive mapper, as SMALT, but your reads should be short (75 bp) , because they were sequenced with HiSeq, in this case I think bowtie 1 is good enough, Once you have your reads mapped, you have to perform a SNP calling. I recommend you freebayes, because it allows to analyze haploid organisms. With your VCF file generated with freeBayes you can perform a functional analysis with SNPeff.

I hope it helps!