struct.error in MACS2 callpeak

Question

[ty0017]

Hi all,

I am trying to callpeaks on sorted and subsampled BAM files from the results of my ChIP-seq program. When I do callpeaks, I get the following error after the read treatment tags step, and I have no idea what it means/how to solve it:

 (macs2_use) [f0036r2@polaris MACS2_R1]$ macs2 callpeak -t /scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam -c /scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_IgG_S10_R1_001_aln_sorted.bam -f BAM -g 1.2e+8 -n SE_def_S12 -B  --outdir /scratch-nfs/f0036r2/FASTQ_R1/BAMout/MACS2_R1
    INFO  @ Sun, 23 May 2021 10:50:27: 
    # Command line: callpeak -t /scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam -c /scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_IgG_S10_R1_001_aln_sorted.bam -f BAM -g 1.2e+8 -n SE_def_S12 -B --outdir /scratch-nfs/f0036r2/FASTQ_R1/BAMout/MACS2_R1
    # ARGUMENTS LIST:
    # name = SE_def_S12
    # format = BAM
    # ChIP-seq file = ['/scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam']
    # control file = ['/scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_IgG_S10_R1_001_aln_sorted.bam']
    # effective genome size = 1.20e+08
    # band width = 300
    # model fold = [5, 50]
    # qvalue cutoff = 5.00e-02
    # The maximum gap between significant sites is assigned as the read length/tag size.
    # The minimum length of peaks is assigned as the predicted fragment length "d".
    # Larger dataset will be scaled towards smaller dataset.
    # Range for calculating regional lambda is: 1000 bps and 10000 bps
    # Broad region calling is off
    # Paired-End mode is off

    INFO  @ Sun, 23 May 2021 10:50:27: #1 read tag files... 
    INFO  @ Sun, 23 May 2021 10:50:27: #1 read treatment tags... 
    struct.error: unpack requires a buffer of 4 bytes
    Exception ignored in: 'MACS2.IO.Parser.BAMParser.tsize'
    struct.error: unpack requires a buffer of 4 bytes
    Traceback (most recent call last):
      File "/dartfs-hpc/rc/home/2/f0036r2/.conda/envs/macs2_use/bin/macs2", line 653, in <module>
        main()
      File "/dartfs-hpc/rc/home/2/f0036r2/.conda/envs/macs2_use/bin/macs2", line 51, in main
        run( args )
      File "/dartfs-hpc/rc/home/2/f0036r2/.conda/envs/macs2_use/lib/python3.6/site-packages/MACS2/callpeak_cmd.py", line 65, in run
        else:       (treat, control) = load_tag_files_options  (options)
      File "/dartfs-hpc/rc/home/2/f0036r2/.conda/envs/macs2_use/lib/python3.6/site-packages/MACS2/callpeak_cmd.py", line 391, in load_tag_files_options
        treat = tp.build_fwtrack()
      File "MACS2/IO/Parser.pyx", line 1169, in MACS2.IO.Parser.BAMParser.build_fwtrack
      File "MACS2/IO/Parser.pyx", line 1181, in MACS2.IO.Parser.BAMParser.build_fwtrack
      File "MACS2/IO/Parser.pyx", line 1166, in MACS2.IO.Parser.BAMParser.get_references
    struct.error: unpack requires a buffer of 4 bytes

Here is the result of samtools quickcheck:

(macs2_use) [f0036r2@polaris MACS2_R1]$ samtools quickcheck -qvvv /scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam
verbosity set to 3
checking /scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam
opened /scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam
/scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam is sequence data
/scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam had no targets in header.
/scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam cannot be checked for EOF block because its filetype does not contain one.
/scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam

And this is the result of samtools flagstat:

(macs2_use) [f0036r2@polaris MACS2_R1]$ samtools flagstat /scratch-nfs/f0036r2/FASTQ_R1/BAMout/BAMsorted/Lee_w_Fe_GFP_2_S12_R1_001_aln_sorted.bam
[E::sam_parse1] missing SAM header
[W::sam_read1] Parse error at line 1
[bam_flagstat_core] Truncated file? Continue anyway.
0 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
0 + 0 mapped (N/A : N/A)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (N/A : N/A)
0 + 0 with itself and mate mapped
0 + 0 singletons (N/A : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

Thanks in advance!

[Ram]

Please use the formatting bar (especially the code option) to present your post better. You can use backticks for inline code (`text` becomes text), or select a chunk of text and use the highlighted button to format it as a code block. If your code has long lines with a single command, break those lines into multiple lines with proper escape sequences so they're easier to read and still run when copy-pasted. I've done it for you this time.

[ty0017]

Thanks! I'm new to all this, so thanks for telling me and fixing the problem.

[ATpoint]

Please add your command line and the output of samtools quickcheck -qvvv your.bam & samtools flagstat your.bam.

[ty0017]

Post updated as requested! Am I correct in believing that the file is actually empty after looking at those outputs?