Miseq adapters

[cookiemic]

Hi, everyone
I am a Miseq newbie
I just got about 1Gb paired end data (150bp X 2) from Miseq
But found that around 8% are adapter contaminations
Some sequences read into the adapter with different degrees, for example:

5' XXXXX(10nt)-ADAPTER(20nt)-SEQUENCES(120nt)
5' XXXXXXXXXXXXX (50nt)-ADAPTER(20nt)-SEQUENCES(80nt)

I don't know if this case is normal. Anyone here can give me some advices?
Thanks in advance.

[kcchan]

What was the size of your inserts? If the size of your library is too short then you will end up sequencing the adapter sequence after the end of the insert.

[pmiguel]

Also, did you make the library? If so, what method/kit did you use?

--
Phillip

[cookiemic]

Thank you for answering my questions.
I am not the one did the wet lab.
So I am confused with the adapter contaminated sequences.
I have asked the wet lab people and waiting for the replies.

[pmiguel]

Okay, I guess 8% is a little high. If I see that many, I want to tighten up my size distribution or do a couple of final ampure cuts prior to running it again. But, although not desirable, it does not necessarily mean there is a big problem.

--
Phillip

[weigrc]

We encounted the similar problem in MiSeq sequencing with samples prepared by Nextera XT kit. If you sequence samples prepared by TruSeq kit, the probelm should not appear...

[pmiguel]

We encounted the similar problem in MiSeq sequencing with samples prepared by Nextera XT kit. If you sequence samples prepared by TruSeq kit, the probelm should not appear...

I don't see why not. Just depends on whether or how you do size selection on a given sample.

--
Phillip

[cookiemic]

However, anyone knows what are those sequence (XXXX) preceding the 20bp of adapter? Thanks.

[csquared]

Adaptors are a lot longer than 20 or 50nt. Are you sure it isn't more adaptor? If it was a truseq kit, looks like you could have gone through the primer and into the index sequence, leading to some diversity in the sequences in that region if there were multiple indexes in the sample sequenced.

[pmiguel]

Adaptors are a lot longer than 20 or 50nt. Are you sure it isn't more adaptor? If it was a truseq kit, looks like you could have gone through the primer and into the index sequence, leading to some diversity in the sequences in that region if there were multiple indexes in the sample sequenced.

I think others have noticed that one of the default protocols for the Miseq causes it to "mask" the adapter sequence, no?
--
Phillip