GS junior amplicon seq - remove adapters

[martin2]

Hi,

does anyone know how I can remove adapters in Roche GS junior sequencing (multiplexed)? I just got the MIDs and primer sequences, but when I look in the fna-file there seems to be more adapter sequence inside.

I developed one tool to find and remove not only MIDs but also primers/adapters/artifacts from any 454 reads (works also on some IonTorrent and Illumina datasets but that's another story). I don't know of any other tool doing that so precisely. The tricky part is how to remove the MIDs on the right side of a read. They are often obfuscated by a number of sequencing or systematic errors and searching for just 10 or 11nt is asking for a trouble. Not even Roche released a software handling the MIDs on the right side (only on the left side). If the software is smart enough it can search for the MID and for the downstream adapter at once taking advantage of longer alignments. Sadly, in the case of multiplexed samples there are lots of combinations to be tested before one can judge what MID and adapter is in the sequence. Seems you have realized there are multiple PCR products stitched together via MID linker ... yes, this happens, quite a lot, not only in amplicon sequencing.

Unfortunately, I offer offer only a commercial data processing service ( http://www.bioinformatics.cz/softwar...rted-protocols ), which does not fullfill your requirements. There is way too much work in it.

[swe5191]

Assembly results of 454 data using Trinity

Hi,
I have performed Trinity denovo assembly using DDBJ pipeline. and I got the following assembly stats as result :
Contig # : 5,009
Total contig size : 2,205,041
Maximum contig size : 3,288
Minimum contig size : 201
N50 contig size : 475

My original data set contained 81146 reads and after preprocessing like quality check and trimming I got 72018 reads.. How do I interpret this result? Is this assembly result a reasonable one?

[silvi]

At least I have some xls report files. I do not understand their output so far.
Sorry for misunderstanding. Of course I just removed the primers and separated the files according to MIDs.
Thanks a lot so far! I will check your recommendations!

[sklages]

ok, you already have SNP tables output by AVA and you want to check the results by some other software ..

Nevertheless you don't want to just remove MID sequences, but separate the data by MID. For the preprocessing I'd still go for SFF Tools.

For the analysis of the amplicons comparable to AVA there are not too many choices ..

Apart from VIP_pipeline I have no idea .. maybe you'll find something here http://omictools.com/

[silvi]

Thanks a lot so far!
The problem is that actually AVA has already been applied (I got ".fna" files to analyze) and the task was to retrieve similar results by a second independent analysis.
As I can see in the data, neither MIDs or primers have been removed yet.

[sklages]

If you just have a few amplicons/exons to cover than AVA is a good option. We use it routinely for smaller "candidate gene projects". As it is also command-line driven, the scripts serve as documentation as well.

Apart from this "SFF Tools" are a good option to deal with SFF in general (MID splitting, file conversions etc.).

[silvi]

Yes that is right. So far I just worked with Illumina and no amlicon data.
So I was also searching for an open-source workflow for variant analysis.

[sklages]

The Roche software can be used for free.

From another post from you I got the impression your are working with amplicon data. Is that correct?

[silvi]

Thanks a lot. Actually I'd like to avoid commercial software or is that for free?
I intended to use flexbar for removal of MID/primer sequences and then STAR to align against the transcriptome.

[sklages]

In general you can freely use all Roche software, in your case the "SFF Tools" for demultiplexing and general SFF file handling, and "GS Amplicon Variant Analyzer (AVA)" software for amplicon analysis. That is still a good option for analyzing amplicons.

AVA has the advantage of being controlled via GUI *or* command line interface.

You just need to fill out the software request form at:

http://454.com/contact-us/software-request.asp

hth,
Sven

[silvi]

Hi,

does anyone know how I can remove adapters in Roche GS junior sequencing (multiplexed)?
I just got the MIDs and primer sequences, but when I look in the fna-file there seems to be more adapter sequence inside.

Thanks for your help!