Question: Paired ends demultiplexing Qiime Illumina
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gravatar for carvin900
5.9 years ago by
carvin9000
Canada
carvin9000 wrote:

Hi, 

sorry in advance for my beginner level in bioinformatic. I need to do analysis of illumina reads paired end. The starting files i have are: fwd.fastq , reverse.fastq and mapping file.txt. 

In the sequences files, there are several samples with different barcodes (barcodes with different lengths) and different primers (3 different set of primers). I tried so far to:

1) I extracted barcodes with extract_barcodes.py --bc1_len X --bc2_len X (where is x is the length of the barcode). I have barcodes of length 4,5,6,7 and 8 so I did 5 times this command with different lengths

2) I merged the reads1.fastq and reads2.fastq output files from 1) for each barcodes length.I had 1,78% in general of non-merging sequences.I also asked to get a fastjoin.join_barcodes.fastq file.

3) I tried to do split_libraries_fastq.py with the merged sequences and the fastjoin.join_barcodes.fastq file. But then I get over 1 900 000 reads non associated to barcodes out of 2 500 000 total reads.

If someone could help me I would be really glad!

Vincent

ADD COMMENTlink modified 5.9 years ago • written 5.9 years ago by carvin9000

Hi Geek_y,

thanks for the rapid answer. I am fairly new in bioinformatic and my research lab is mostly using Qiime, so haven't heard of CASAVA. Also, our local server is based on Qiime. Would it be possible to launch commands from Qiime to CASAVA? Thanks for your help!

Vincent

 

ADD REPLYlink written 5.9 years ago by carvin9000

Thanks for your help Geek_y, i found out my mistake. There was a switch of two bases in our primer sequence registered and the primer sequence in the reads. Now all is doing fine. I only have done merging and already split_libraries.py. Thanks again!

ADD REPLYlink written 5.9 years ago by carvin9000
1
gravatar for geek_y
5.9 years ago by
geek_y11k
Barcelona
geek_y11k wrote:

Why don't you use illumina cassava for demultiplexing the data ? Is it a GBS kind of data where you have internal barcodes other than standard barcodes used for multiplexing ?

ADD COMMENTlink written 5.9 years ago by geek_y11k
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