SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
Genomic DNA sample preparation Smriti Sample Prep / Library Generation 2 05-25-2012 01:09 PM
PubMed: ShoRAH: estimating the genetic diversity of a mixed sample from next-generati Newsbot! Literature Watch 0 09-20-2011 02:00 AM
Mixed sample and starting amount questions vepached64 General 2 05-30-2011 05:46 AM
Sample/library prep of DNA and RNA in a metagenomic sample chrisaw01 Metagenomics 1 05-05-2011 01:59 PM
Can smRNA sequencing primer and genomic DNA sequencing primer mixed in one lane? whimsy Illumina/Solexa 3 04-06-2011 09:30 AM

Reply
 
Thread Tools
Old 09-15-2011, 01:17 AM   #1
rubi
Junior Member
 
Location: North Carolina

Join Date: Apr 2011
Posts: 6
Default Assembling a mixed DNA sample

Hi all,
I sequenced (Illumina GAII, 75 bp paired end reads, average insert 250) genomic DNA from a sample which contains both a host's and its parasite's DNA, where it is enriched with the parasite's DNA.

I'm only interested in assembling the parasite's genome, and it is a non reference genome so I'm doing denovo assembly.

As a filtering step I removed any of the reads that mapped to the host's EST data (there's no reference genome foe the host either), but that probably only removed protein coding DNA.

So I'm still stuck with non-coding DNA from the host, which I would like to remove. My thought was that since the reads are enriched with the parasite's DNA, contigs originating from the host should have low coverage.

The problem is that the assembly, produced using SOAPdenovo, uses Kmers to produce contigs and the result is that 80% of the contigs are as 100 bp short and no read maps to them so they have 0 coverage.

So my question is how do I work around this problem?
Should I use a larger Kmer size? Should I assume that these short contigs indeed represent the host's non-coding DNA and just filter them?

Thanks

Last edited by rubi; 09-15-2011 at 07:23 AM.
rubi is offline   Reply With Quote
Old 10-21-2011, 06:17 AM   #2
FredericRaymond
Junior Member
 
Location: Canada

Join Date: Oct 2011
Posts: 8
Default

Hello,

Have you tried aligning the reads to the human genome to filter the reads that are from the host? Afterwards, you could counterverify the filtered reads by aligning them to related parasite genomes to make sure you do not miss some parasite sequences.

In addition, you may want to try the Ray assembler, which also uses K-mers and performs very well with paired data. The use of this assembler may even solve part of you problem, since Ray considers the paired-read context to created contigs from the k-mer-based graph.

FR
FredericRaymond is offline   Reply With Quote
Old 10-21-2011, 08:33 AM   #3
HESmith
Senior Member
 
Location: Bethesda MD

Join Date: Oct 2009
Posts: 503
Default

If the host-parasite relationship is not obligate, you could sequence just the host genome, assembly it, and use it to filter your existing data. Read depth would serve as an independent validation of this strategy.
HESmith is offline   Reply With Quote
Old 11-24-2011, 02:53 PM   #4
pytheus
Junior Member
 
Location: UK

Join Date: May 2011
Posts: 2
Default

If you're studying something with exteme's of GC content like Plasmodium falciparum (~20%) you might be able to discriminate on GC content.
pytheus is offline   Reply With Quote
Reply

Tags
assembly, contigs, coverage, read

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 08:57 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO