Adapter removal...

[tjten]

Hello,

First off I wanted to thank the seqanswers community for being a very valuable resource. I'm a graduate student new to NGS and have learned a ton from this community.

So my lab is attempting to assemble a fungal genome believed to be around 40Mb in size. We had our DNA sample sequenced commercially and received 24 million paired reads of high quality (according to the FastQC) report.

This is our first genome assembly project so we are going through a bit of a learning curve. My question concerns the adapters used in Illumina sequencing technology. The company used a HiSeq2000 to generate our reads. Do we need to remove the adapters from these reads before we attempt assembly? How would I know if the adapters are present in the reads? Just looking at them doesn't reveal a common sequence, and the FastQC report does not report any 'over represented' sequences. Is this enough evidence to conclude that the adapters are not present in the reads? Are they normally even included? Thanks in advance for your responses.

[shunyip]

If FastQC couldn't find any adapters, they might had been trimmed for you. However, if you have a HCC, it doesn't hurt to trim them on your own just in case, since trimming is a pretty fast step. Also, you can filter out low quality reads while you are at it. Personally, I use Trimmomatic to do that.

Edit: if you received raw reads, yes, adapter sequences should be present in your reads. In this case, you will need to remove the adapters first.

[tjten]

I'm assuming they are raw reads, but honestly I'm not sure... is it customary for commercial sequencing to trim the adapters they used off of the reads? The only thing that makes me think the adapters are already trimmed is that the FastQC report doesn't pick any up, which you would think it would.

I looked into the program you suggested. I noticed it includes the TruSeq adapters, but what if a different kit was used. Would we just have to to request the adapter sequences from the company and feed these into trimmomatic?

I was also thinking about just trimming either end of each read since our coverage is fairly high anyway. Would that approach work to remove any lingering adapters? How long is a typical adapter, and could it be on either end or just the beginning of the read?

Sorry for so many questions, I do appreciate your help.

[shunyip]

A Quote from trimmomatic's website:
"Illumina adapter and other technical sequences are copyrighted by Illumina,but we have been granted permission to distribute them with Trimmomatic. Suggested adapter sequences are provided for TruSeq2 (as used in GAII machines) and TruSeq3 (as used by HiSeq and MiSeq machines), for both single-end and paired-end mode. These sequences have not been extensively tested, and depending on specific issues which may occur in library preparation, other sequences may work better for a given dataset."

Assuming that the results you have are raw reads and you didn't pay extra for them to trim them for you, they should have adapter sequences in them.

Yes, to remove adapters, you need to know their sequences beforehand. Trimmomatic has provided several adapter sequences with their software (under the /adapters folder). So, if you know which machine you used for sequencing (you should), just choose the right adapter file and it will be fine. Otherwise, yes, it is best to contact the sequencing company for more info.

To get an idea about the adapters, you can open the adapter files in Trimmomatic with your text editor. Also, if you are using FastQC, there is a text file under the "Contaminants" folder which contains some trimmed adapters.

Adapters appear on both ends of your reads. Well, so many tools are now available to trim adapters, so it is not advised to just blindly trim both ends of your reads.


No problem , I hope this helps,

[ctseto]

Search google for "illumina adapter letter"

http://support.illumina.com/downloads/illumina_adapter_sequences_letter.ilmn

Depending on what you are running, check and see if the adapters used in your workflow are represented in the various adaptor files that come with Trimmomatic.

[GenoMax]

This question is most probably cross-posted and discussed in this thread (same user but different date/posting): http://seqanswers.com/forums/showthread.php?t=40133