Demultiplexing nanopore reads

Question

[kirillkirilenko]

Hey everyone!

My problem is next: I got many FAST5 files from MiniON, then I run guppy to basecall them (with default parameters on GPU). As a result I got FastQ-files. Then I read there were a lot of ways for demultiplexing my reads. And I chose Qcat, it gave me 18 FastQ-files each from different experiment (they differ in barcodes). And now I can't understand my reads trimmed or not? I've tried to run FastQC with one of these files and I didn't see overrepresented sequencies or adapters. May be in general problem is: do demultiplexing trim the reads while defining their barcodes? Btw I ran Qcat without additional parameters.

[GenoMax]

Use porechop if you are looking to trim the reads: Demultiplexing MinION reads

[Roland]

Hi I have a fastq file from guppy base caller and am trying to demultiplex with porechop, and I get an error saying that Porechop could not determine the barcode orientation. This is the the code I ran. Can someone help me understand this error? Thank you.

rasika@CPHD-NGS:~/CTNG_data$ porechop -i rasika_merged.fastq -b CTNG_out
Loading reads
rasika_merged.fastq
66,511 reads loaded
Looking for known adapter sets
10,000 / 10,000 (100.0%)

Error: Porechop could not determine barcode orientation

[GenoMax]

Please create a new question for this and then delete this post. Your question is not related to the original one here.

Answer 1

[Carambakaracho]

I'm not much of a nanopore nor Qcat expert, but demultiplexing with Qcat does not trim the reads, according to its manual

[Prakki Rama]

But below in the "full usage" section, there is a parameter to trim the reads. Do not know if the documentation is not updated.

--trim                Remove adapter and barcode sequences from reads.

In any case, the tool is now not recommended for the usage.