How to truncate fastq files before fastq-join?

[birgitte.moen]

Hi,

 

We use the paired-end 16S rDNA amplicon designed by Caporaso et al., and have previously sequenced 2x150bp using the Illumina MiSeq V2 kit. Now we wanted to try the V3 kit and sequenced 301 cycles.....Taken into account that the total lenght of the amplicon is approx. 250bp that was a mistake, as it lookes like many of the output sequences are longer that the amplicon (nonsense information at the end?) 

As a result when we try the fastq-join script i QIIME we only get a few sequences out.

 

Is there a command I can use in Qiime to truncate Read1 og Read2 to for example 200bp before fast-join?

 

(Aslo, is there anything to gain using the V3 kit for this amplicon? And if so, what is the optimum number of cycles?)

 

Kind regards

Birgitte

[Erik Lysøe]

Hi

Install seqtk (https://github.com/lh3/seqtk) and use "./seqtk trimfq -e xx in.fastq > out.fastq" to remove xx number of bp at the end

Cheers

Erik

[Tony Walters]

Thank you for the help Erik. I also received some advice from Mike Robeson:

"Yeah, I guess the extra sequence at the end is creating to many mismatches where the reads overlap. Not much that can be done other than trimming the fastq files. 

The quickest and easiest way would be to use the latest usearch to simply trim the forward and reverse reads separately:

usearch -fastq_filter fw_seqs.fastq -fastqout trunc_fw_seqs.fastq -fastq_trunclen 200

Repeat for the reverse reads, and then join the pairs. I do not think any other filtering is performed with this command."

--
 
---
You received this message because you are subscribed to the Google Groups "Qiime Forum" group.
To unsubscribe from this group and stop receiving emails from it, send an email to qiime-forum...@googlegroups.com.
For more options, visit https://groups.google.com/groups/opt_out.

[Erik Lysøe]

Hi

An even easier solution (for biologists) is to use mothur (http://www.mothur.org/wiki/Download_mothur), the first command in the miseq sop (http://www.mothur.org/wiki/MiSeq_SOP)

make.contigs(file=stability.files, trimoverlap=T, processors=4)

Look at the example how a stability file look like

The reads are running through the primers and causing problems (with 300bp), and trimoverlap will trim the reads to overlap the same bases. As I understand it, similar to fastq-join

I use mothur all the way to shared file, and then make a biom file (http://www.mothur.org/wiki/Make.biom), that I use to make figures in qiime. But I guess it's just to take the resulting fasta from make.contigs and continue in qiime

Cheers

Erik

[idamoverleir]

Hi Erik,

Do I have to sign up to be able to download seqtk using your link?

Thanks