problem with trimming with trim-galore

[Brawni]

Hello!

Thanks again for sharing data and code! Im working through your script to process scATAC-seq fastq files and Im encountering some errors when I trim the demultiplexed fastq files using trim-galore with the following code:

for sample in $(cat $sample_ID); do echo trimming $sample; trim_galore $demultiplex_folder/$sample*R1*.gz $demultiplex_folder/$sample*R2*.gz --paired -a CTGTCTCTTATA -a2 CTGTCTCTTATA --three_prime_clip_R1 1 --three_prime_clip_R2 1 -o $trimmed_fastq ; done
echo "All trimmed file generated."

It looks like the trimming works well up to the last 8 files in the sample_ID.txt where trim-galore crashes reporting different errors. Please find attached the error reports files.
In the majority of the cases errors refer to a problem with a corrupt or truncated input files so I re-downloaded the fastq-files and demultiplexed them a second time but the errors are still there.

Im working on a conda environment with python 2.7, cutadapt=1.8.3 and trim-galore=0.4.1.
ATTCTTAGCT.ACTCTCTCAA.R1.fastq.gz_trimming_report.txt
ATTCTTAGCT.GTCGCTCAGT.R1.fastq.gz_trimming_report.txt
ATTCTTAGCT.TGATTCCTCA.R1.fastq.gz_trimming_report.txt
ATTCTTAGCT.CCGTCGGAGG.R1.fastq.gz_trimming_report.txt
ATTCTTAGCT.AGCGTCAATG.R1.fastq.gz_trimming_report.txt
ATTCTTAGCT.TTGAGTATCA.R1.fastq.gz_trimming_report.txt
ATTCTTAGCT.TGAGGTAGAA.R1.fastq.gz_trimming_report.txt

Any suggestions on what the cause might be?

Much appreciated!!

[JunyueC]

Hi, We use the same version of cut adapt and trim-galore and haven’t met this problem before. Could you check if you can unzip the fastq flies to see if the files are really truncated?

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On Aug 31, 2020, at 6:36 PM, Bruno Giotti ***@***.***> wrote: Hello! Thanks again for sharing data and code! Im working through your script to process scATAC-seq fastq files and Im encountering some errors when I trim the demultiplexed fastq files using trim-galore with the following code: for sample in $(cat $sample_ID); do echo trimming $sample; trim_galore $demultiplex_folder/$sample*R1*.gz $demultiplex_folder/$sample*R2*.gz --paired -a CTGTCTCTTATA -a2 CTGTCTCTTATA --three_prime_clip_R1 1 --three_prime_clip_R2 1 -o $trimmed_fastq ; done echo "All trimmed file generated." It looks like the trimming works well up to the last 8 files in the sample_ID.txt for which trim-galore crashes reporting different errors. Please find attached the error reports files. As it says the files might be truncated I re-downloaded the fastq-files and demultiplexed them a second time but the errors are still there. Im working on a conda environment with python 2.7, cutadapt=1.8.3 and trim-galore=0.4.1. ATTCTTAGCT.ACTCTCTCAA.R1.fastq.gz_trimming_report.txt <https://github.com/JunyueC/sci-CAR_analysis/files/5152930/ATTCTTAGCT.ACTCTCTCAA.R1.fastq.gz_trimming_report.txt> ATTCTTAGCT.GTCGCTCAGT.R1.fastq.gz_trimming_report.txt <https://github.com/JunyueC/sci-CAR_analysis/files/5152931/ATTCTTAGCT.GTCGCTCAGT.R1.fastq.gz_trimming_report.txt> ATTCTTAGCT.TGATTCCTCA.R1.fastq.gz_trimming_report.txt <https://github.com/JunyueC/sci-CAR_analysis/files/5152932/ATTCTTAGCT.TGATTCCTCA.R1.fastq.gz_trimming_report.txt> ATTCTTAGCT.CCGTCGGAGG.R1.fastq.gz_trimming_report.txt <https://github.com/JunyueC/sci-CAR_analysis/files/5152933/ATTCTTAGCT.CCGTCGGAGG.R1.fastq.gz_trimming_report.txt> ATTCTTAGCT.AGCGTCAATG.R1.fastq.gz_trimming_report.txt <https://github.com/JunyueC/sci-CAR_analysis/files/5152934/ATTCTTAGCT.AGCGTCAATG.R1.fastq.gz_trimming_report.txt> ATTCTTAGCT.TTGAGTATCA.R1.fastq.gz_trimming_report.txt <https://github.com/JunyueC/sci-CAR_analysis/files/5152935/ATTCTTAGCT.TTGAGTATCA.R1.fastq.gz_trimming_report.txt> ATTCTTAGCT.TGAGGTAGAA.R1.fastq.gz_trimming_report.txt <https://github.com/JunyueC/sci-CAR_analysis/files/5152936/ATTCTTAGCT.TGAGGTAGAA.R1.fastq.gz_trimming_report.txt> Any suggestions on what the cause might be? Much appreciated!! — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#4>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ADSB5ODZN52553KRDXKGZPDSDQQW5ANCNFSM4QRAJ5KA>.

[Brawni]

Thanks for your reply.
I tried unzip as suggested and it seems that the problem is affecting several fastq files. This is a snapshots of the kind of error I'm getting when I try to unzip them.

Im unzipping them like this

for sample in $(cat $sample_ID); 
	do 
	gunzip -c $demultiplex_folder/$sample.R1.fastq.gz > $unzipped_folder/$sample.R1.fastq
	gunzip -c $demultiplex_folder/$sample.R2.fastq.gz > $unzipped_folder/$sample.R2.fastq
	done

Thanks for your feedbacks!

[JunyueC]

Hi Bruno, I checked the original data and it is fine: https://shendure-web.gs.washington.edu/content/members/cao1025/public/nobackup/sciCAR_ATAC_fastq/ <https://shendure-web.gs.washington.edu/content/members/cao1025/public/nobackup/sciCAR_ATAC_fastq/> The problem is potentially due to the demultiplexing step instead of the trimming step? -I would suggest you check if there are any error messages during demultiplexing. Jun

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On Sep 1, 2020, at 1:12 PM, Bruno Giotti ***@***.***> wrote: Thanks for your reply. I tried unzip as suggested and it seems that the problem is affecting several fastq files. This is a snapshots of the kind of error I'm getting when I try to unzip them. <https://user-images.githubusercontent.com/58828216/91884729-9508a080-ec54-11ea-8815-3ca1a28df51d.png> Im unzipping them like this for sample in $(cat $sample_ID); do gunzip -c $demultiplex_folder/$sample.R1.fastq.gz > $unzipped_folder/$sample.R1.fastq gunzip -c $demultiplex_folder/$sample.R2.fastq.gz > $unzipped_folder/$sample.R2.fastq done Thanks for your feedbacks! — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#4 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ADSB5OEINRO5HWDVFIQF6YTSDUTRPANCNFSM4QRAJ5KA>.