erinyoung
commented
1 year ago Oh no!
This is from https://github.com/lh3/bwa/issues/228#issuecomment-534769169 , which may apply here
If bwa is terminated with this error, it usually signifies that pair reads in both FASTQ files are disordered.
From BBTools Repair Guide:
With paired reads in 2 files, the first read in file 1 must be the mate of the first read in file 2, etc. For paired reads in a single interleaved file, the second read is the mate of the first read, and the 4th read is the mate of the 3rd read, etc.
There are few solutions which worked for me.
Sort FASTQ files
BBTools repair.sh is supposed to do that, but it seems it cannot fix large datasets as it loads whole file to RAM.
FASTQ-SORT is way better for large FASTQs. It buffers RAM on hard drive, it successfully sorted ~ 150GB file.
Use another aligner
Bowtie 2 doesn't require the condition of mates in the same order, but while aligning disordered FASTQs with Bowtie 2 (as of my experience) you get less mapped reads.So you can try to repair your fastq files with bbtools' repair.sh or trying use minimap for alignment (set params.aligner = 'minimap2')
DrB-S
I am running the latest version of Cecret on paired-end fastqs as follows: nextflow run UPHL-BioNGS/Cecret -profile singularity -c configs/sarscov.config --medcpus 70 --maxcpus 100 --reads reads --relatedness true --msa nextalign
The reads files all end with _R(1,2}.fastq A typical paired name is: 2023CV1635.BC4L11.2023-06-29.01.C03_R1.fastq and 2023CV1635.BC4L11.2023-06-29.01.C03_R2.fastq
They were derived from an interleaved fastq file (2023CV1635.BC4L11.2023-06-29.01.C03.fastq.gz) after unzipping using the following:
fastaq deinterleave 2023CV1635.BC4L11.2023-06-29.01.C03.fastq 2023CV1635.BC4L11.2023-06-29.01.C03_R1.fastq 2023CV1635.BC4L11.2023-06-29.01.C03_R2.fastq
Here is the error: ERROR ~ Error executing process > 'CECRET:cecret:bwa (2023CV1635.BC4L11.2023-06-29.01.C03)'
Caused by: Process
CECRET:cecret:bwa (2023CV1635.BC4L11.2023-06-29.01.C03)terminated with an error exit status (1)Command executed:
mkdir -p bwa logs/CECRET:cecret:bwa log=logs/CECRET:cecret:bwa/2023CV1635.BC4L11.2023-06-29.01.C03.2e441589-1655-44d3-8de3-d34884116268.log
time stamp + capturing tool versions
date > $log echo "bwa $(bwa 2>&1 | grep Version )" >> $log bwa_version="bwa : "$(bwa 2>&1 | grep Version)
index the reference fasta file
bwa index MN908947.3.fasta
bwa mem command
bwa mem -t 100 MN908947.3.fasta 2023CV1635.BC4L11.2023-06-29.01.C03_clean_PE1.fastq.gz 2023CV1635.BC4L11.2023-06-29.01.C03_clean_PE2.fastq.gz > bwa/2023CV1635.BC4L11.2023-06-29.01.C03.sam
Command exit status: 1
Command output: (empty)
Command error: [bwa_index] Pack FASTA... 0.00 sec [bwa_index] Construct BWT for the packed sequence... [bwa_index] 0.01 seconds elapse. [bwa_index] Update BWT... 0.00 sec [bwa_index] Pack forward-only FASTA... 0.00 sec [bwa_index] Construct SA from BWT and Occ... 0.00 sec [main] Version: 0.7.17-r1188 [main] CMD: bwa index MN908947.3.fasta [main] Real time: 0.028 sec; CPU: 0.017 sec [M::bwa_idx_load_from_disk] read 0 ALT contigs [M::process] read 3830 sequences (550465 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (505, 0, 0, 501) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (709, 3832, 7080) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 19822) [M::mem_pestat] mean and std.dev: (4089.34, 3196.52) [M::mem_pestat] low and high boundaries for proper pairs: (1, 26193) [M::mem_pestat] skip orientation FR as there are not enough pairs [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation RR... [M::mem_pestat] (25, 50, 75) percentile: (1059, 3892, 7015) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 18927) [M::mem_pestat] mean and std.dev: (4197.31, 3100.22) [M::mem_pestat] low and high boundaries for proper pairs: (1, 24883) [mem_sam_pe] paired reads have different names: "00056560-4cc8-4869-a2f5-855b469d586d", "001c0b61-b674-4486-81fa-7ffac2957da1"
Work dir: /data/Sequence_analysis/Cecret/Analyses/Covid_Terra_6Sep23/work/33/d5997c0f46ce6f7cdd83b12ec362af
=== NOTE: The same error occurred when I did not deinterleave the files first!