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valenlab / amplican

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There are no events. Check whether you have correct primers in the config file. #17

Open colin893 opened 5 months ago

colin893 commented 5 months ago

Hi,

Thanks for the tool. I wanted to run the pipeline to detects HDR at given position based on sequencing results on zebrafish larvae after CrisperCas9 experiments. I followed the recommendations and looked at the example files to design my config.csv.

First, can we use fasta.gz files as input?

Second, when I run the pipeline, here is what I get :

Checking write access... Checking configuration file... Making alignments... Aligning reads for IC-5412-11-0606-35e-B Aligning reads for IC-5412-11-0606-50e Saving alignments... Saving parameters... Saving unassigned sequences... Saving barcode statistics... Translating alignments into events... Saving complete events - unfiltered... Error in amplicanPipeline(paste(getwd(), "config.csv", sep = "/"), paste(getwd(), : There are no events.Check whether you have correct primers in the config file.

I have looked at the primers sequences and compared with the ones used in examples, found nothing weird. I have to say that I am not at all experienced in such experiments, so I can be missing a critical point. All I know is that Crispresso outputs results with the same inputs.

I get empty file for alignement in results, so I guess something going wrong in there?

If you can have a look at the barcodeReadFilter.txt file :

Barcode experiment_count read_count bad_base_quality bad_average_quality bad_alphabet filtered_read_count unique_reads unassigned_reads assigned_reads IC-5412-11-0606-35e-B 1 14250 0 722 0 13528 1774 1774 0 IC-5412-11-0606-50e 1 15859 0 1009 0 14850 2631 2631 0

Would you have an idea? Thanks in advance for your time,

JokingHero commented 5 months ago

Amplican supports gzipped FASTQ, but not FASTA. Transform your FASTA to FASTQ first, you can use somthing like seqkit for that.

Next, I see you have 0 assigned reads, so something is wrong with how reads are being split by barcode, for example forward primer and reverse primer should be there.

You should check the top alignments of your reads listed in the Barcode report. Do these alignments contain proper primers?