I run follow Command-line code:

Linux (Ununtu 22.04.5 LTS in Whidows WSL)

hisat2 -p 6 --dta -x /mnt/d/hiv_omics/scripts/Hisat2_index/genome_tran  -U /mnt/d/hiv_omics/GSE180044/SRR15127754/trimmed_output/SRR15127754_trimmed.fq.gz  -S /mnt/d/hiv_omics/GSE180044/SRR15127754/alignment/SRR15127754_align.sam

samtools view -@ 8 -bS /mnt/d/hiv_omics/GSE180044/SRR15127754/alignment/SRR15127754_align.sam | samtools sort -@ 8 -o /mnt/d/hiv_omics/GSE180044/SRR15127754/alignment/SRR15127754_align.sorted.bam

samtools markdup -@ 8 -r -s /mnt/d/hiv_omics/GSE180044/SRR15127754/alignment/SRR15127754_align.sorted.bam /mnt/d/hiv_omics/GSE180044/SRR15127754/alignment/SRR15127754_align.dedup.bam

featureCounts -T 8 -t exon -g gene_id -a /mnt/d/hiv_omics/scripts/Homo_sapiens.GRCh38.114.gtf --primary -o /mnt/d/hiv_omics/GSE180044/SRR15127754/alignment/SRR15127754_counts.txt /mnt/d/hiv_omics/GSE180044/SRR15127754/alignment/SRR15127754_align.dedup.bam

In the final step (featureCount), the report error is: buffer overflow detected : terminated Aborted (core dumped)

How can I solve it. Thanks. Rui Tang

Hello Rui Tang,

The buffer overflow error in featureCounts that you encountered is likely due to a compatibility issue between the Subread package and the glibc library in your WSL environment. This can trigger memory handling problems, especially with large BAM files or annotation data, leading to the detected overflow and core dump.

To resolve this, first verify your featureCounts version by running:

featureCounts -v

If it is older than 2.0.0, update to the latest Subread package (currently version 2.1.1 is available via Bioconda). Install Bioconda if needed, then use:

conda install -c bioconda subread

If the error persists, recompile Subread from source with an updated glibc. Update glibc in your Ubuntu WSL to at least version 2.35 (which is default in 22.04, but confirm with ldd --version). Download Subread source from https://subread.sourceforge.net/, extract it, and compile:

cd src
make -f Makefile.Linux

Add the bin directory to your PATH:

export PATH=$PATH:/path/to/subread/bin

Additionally, WSL performance degrades on Windows-mounted drives like /mnt/d. Copy your files to a Linux directory such as ~/data and rerun the pipeline from there to avoid I/O bottlenecks that may contribute to the error.

Test with fewer threads initially:

featureCounts -T 1 -t exon -g gene_id -a /path/to/Homo_sapiens.GRCh38.114.gtf --primary -o /path/to/SRR15127754_counts.txt /path/to/SRR15127754_align.dedup.bam

If successful, increase threads gradually. Also, validate your BAM file:

samtools quickcheck /path/to/SRR15127754_align.dedup.bam

If it reports issues, recreate the BAM.

Kevin