trim seqs

Bjcampbell · December 17, 2013, 1:43pm

I have MiSeq data that was successfully grouped with the make.contigs file. However, I want to use trim.seqs to get rid of the primers, but it keeps putting most of the sequences in the scrap file. I know this is because the primers are not at the start of the sequence. Is there a way I can use trim sequences to search for the primer and get rid of all the sequences prior to that? Or, do I have to put in all the barcode information (I used the double barcodes as in the MiSeq paper by Kozich et al, 2013). If so, what is the best way of doing that that includes the barcodes, spacer and primers? It is not specifically listed in the trim.seqs command information. Thanks!

pschloss · December 17, 2013, 3:47pm

If you followed the approach we used in Kozich, there won’t be any barcodes or primers on the sequences and so trim.seqs won’t be necessary. If you aren’t following the MiSeq wet lab SOP that we posted, can you give some more details of what you’ve done?

Bjcampbell · December 17, 2013, 7:06pm

I emailed you back in November and ended up getting instructions from Jim Kosich about setting up a sample sheet. However, the sequencing provider could not make the sample sheet work last time (miseq rejected it) so they demultiplexed the run afterward. So, the sequences were already split into samples when I got the data, and I then created a stability file based on the names. They have the barcode, linker and primers still in the sequences. I would like to remove them so I can work with a smaller number of sequences- right now it is at 3.5 million, with only 22 samples (out of 96). Any suggestions would be appreciated.

westcott · December 18, 2013, 2:11pm

Could you post a sample sequence with the barcode, linker and primer?

jayt · December 30, 2013, 4:06am

I’m also curious about this. I am following the Miseq tutorial and I am not sure what primers were used, if there are any adapters/barcodes/linkers on the sequences, and if the make.configs command does this automatically. I also cannot find the paper that was mentioned as being published in AEM. I’d like to use the tutorial to test the abilities of a cloud based tool called CloVR. It would be really nice if I could get the results for the sample test or the entire test. Please help!

pschloss · January 6, 2014, 8:31pm

ncbi.nlm.nih.gov

Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.

JJ Kozich, SL Westcott, NT Baxter, SK Highlander and PD Schloss, Applied and environmental microbiology, Sep 2013

Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.

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trim seqs

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