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rrwick / Porechop

adapter trimmer for Oxford Nanopore reads
GNU General Public License v3.0
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Working with Custom barcodes #60

Open cwachtel opened 6 years ago

cwachtel commented 6 years ago

Hi,

I am trying to work with Porechop to demultiplex my data. The libraries are a bit complex, and I would really appreciate some help here. We are doing microbiome work, and our hoping to do 16s amplicon sequencing of the full length 16s gene. We have used the Nanopore kit, but we would like to increase the number of samples we use per run, which requires us to use our own primers and barcodes.

We have 16s primers for the full length gene that we added the Common sequences used by Fluidigm to the tails. We then did a second PCR with the Fluidigm primers, which adds index and adaptor (for illumina) sequnecs to the the samples. We pooled the samples and then added the Nanopore adaptors using the Ligation Sequencing kit 1D

I have followed the instructions within adaptors.py to add the barcode sequences for fluidigm, but when I run the demultiplexing, the indexes are not found. I have looked at the FASTQ files manually and I can find the sequences, I just cant figure out how to correctly set up the adaptor.py file so that it will properly identify the barcode sequence.

Any help would be greatly appreciated.

Thanks Chaim

ieknot commented 4 years ago

Hi, this will probably be irrelevant for you by now, but I'm replying for anyone else that might experience this same problem.

I just solved this problem by uninstalling Porechop (simply remove the folder from your computer). Then I cloned the Porechop git again (as the first step of the installation procedure dictated), but before assembling it (the next 2 lines in the installation code), I added my primers to the the adapter.py file. After I saved the file, I completed the installation. Don't know whether I'll have to keep doing this whenever I want to add new primers, but works for now!