PBAT dataset failed in calling step

[ifishlin]

Hi, I used the config files on UserGuide(includes IHEC_standard.conf and IHEC_PBAT.conf) to run the gemBS on a PBAT datasets. But it failed in the calling step, and I can't tell what was wrong from the log message.

I tried both the v3.5.1_IHEC branch (built by myself) and the docker "heathsc/gembs:latest", both give me the same results.

Is there any suggestion? Thanks

: Command map started at 2021-01-19 22:05:43.782236
:
: ------------ Mapping Påarameters ------------
: Sample barcode    : pbat_data
: Data set          : pbat_data
: No. threads       : 24
: Index             : indexes/hg_GRCh38_PhiX_Lambda.BS.gem
: Paired            : True
: Read non stranded : True
: Reverse conversion: False
: Type              : PAIRED
: Input Files       : /benchmark_data/pbat/pbat_data/fastq/pbat_data_trimmed_R1.fastq.gz,/benchmark_data/pbat/pbat_data/fastq/pbat_data_trimmed_R2.fastq.gz
: Output dir        : ./mapping/pbat_data
:
: Bisulfite Mapping...
: Bisulfite Mapping done. Output File: /benchmark_data/gembs_pbat/mapping/pbat_data/pbat_data.bam
: Merging process done for pbat_data. Output files generated: /benchmark_data/gembs_pbat/mapping/pbat_data/pbat_data.csi
:
: Command call started at 2021-01-20 05:48:25.203063
:
: ----------- Methylation Calling --------
: Reference       : indexes/hg_GRCh38_PhiX_Lambda.gemBS.ref
: Species         : Human
: Right Trim      : 0
: Left Trim       : 5
: Chromosomes     : ['chr1', 'chr2', 'chr3', 'chr4', 'chr5', 'chr6', 'chr7', 'chrX', 'chr8', 'chr9', 'chr11', 'chr10', 'chr12', 'chr13', 'chr14', 'chr15', 'chr16', 'chr17', 'chr18', 'chr20', 'chr19', 'chrY', 'chr22', 'chr21', '@pool_1']
: Threads         : 24
: Sample: pbat_data    Bam: ./mapping/pbat_data/pbat_data.bam
:
: Methylation Calling...
2021-01-20 05:49:02,167 ERROR: Process '/usr/local/lib/python3.8/dist-packages/gemBS/gemBSbinaries/bs_call' finished with -6
2021-01-20 05:49:02,168 ERROR: Additional threads:  10 7 7
2021-01-20 05:49:02,168 ERROR: Loading reference sequence index
2021-01-20 05:49:02,168 ERROR: Sequence index read in successfully
2021-01-20 05:49:02,168 ERROR: Opened ./mapping/pbat_data/pbat_data.bam for input (Index)
2021-01-20 05:49:02,168 ERROR: Processing region chr4:1-190214555
2021-01-20 05:49:02,168 ERROR: Processing chromosome chr4 (OK)
2021-01-20 05:49:02,168 ERROR: Loading reference for chr4
2021-01-20 05:49:02,168 ERROR: len = 190214555
2021-01-20 05:49:02,168 ERROR: Read in 190204555 bases on chr4 from 10001 - 190214555, stored in 76081824 bytes
2021-01-20 05:49:02,168 ERROR: bs_call: get_template_vector.c:239: read_input: Assertion `al->forward_position == thash->al->forward_position && al->reverse_position == thash->al->reverse_position' failed.
Exception in thread Thread-4:
ValueError: Error while executing the bscall process.
2021-01-20 05:49:37,257 ERROR: Process '/usr/local/lib/python3.8/dist-packages/gemBS/gemBSbinaries/bs_call' finished with -6
2021-01-20 05:49:37,258 ERROR: Additional threads:  10 7 7
2021-01-20 05:49:37,258 ERROR: Loading reference sequence index
2021-01-20 05:49:37,258 ERROR: Sequence index read in successfully
2021-01-20 05:49:37,258 ERROR: Opened ./mapping/pbat_data/pbat_data.bam for input (Index)
2021-01-20 05:49:37,258 ERROR: Processing region chr2:1-242193529
2021-01-20 05:49:37,258 ERROR: Processing chromosome chr2 (OK)
2021-01-20 05:49:37,258 ERROR: Loading reference for chr2
2021-01-20 05:49:37,258 ERROR: len = 242193529
2021-01-20 05:49:37,258 ERROR: Read in 242183529 bases on chr2 from 10001 - 242193529, stored in 96873414 bytes
2021-01-20 05:49:37,258 ERROR: bs_call: get_template_vector.c:239: read_input: Assertion `al->forward_position == thash->al->forward_position && al->reverse_position == thash->al->reverse_position' failed.
Exception in thread Thread-2:
ValueError: Error while executing the bscall process.
2021-01-20 05:49:45,471 ERROR: Process '/usr/local/lib/python3.8/dist-packages/gemBS/gemBSbinaries/bs_call' finished with -6
2021-01-20 05:49:45,472 ERROR: Additional threads:  10 7 7
2021-01-20 05:49:45,472 ERROR: Loading reference sequence index
2021-01-20 05:49:45,472 ERROR: Sequence index read in successfully
2021-01-20 05:49:45,472 ERROR: Opened ./mapping/pbat_data/pbat_data.bam for input (Index)
2021-01-20 05:49:45,472 ERROR: Processing region chr1:1-248956422
2021-01-20 05:49:45,472 ERROR: Processing chromosome chr1 (OK)
2021-01-20 05:49:45,472 ERROR: Loading reference for chr1
2021-01-20 05:49:45,472 ERROR: len = 248956422
2021-01-20 05:49:45,472 ERROR: Read in 248946422 bases on chr1 from 10001 - 248956422, stored in 99578572 bytes
2021-01-20 05:49:45,472 ERROR: bs_call: get_template_vector.c:239: read_input: Assertion `al->forward_position == thash->al->forward_position && al->reverse_position == thash->al->reverse_position' failed.
Exception in thread Thread-1:
ValueError: Error while executing the bscall process.
2021-01-20 05:51:18,764 ERROR: Process '/usr/local/lib/python3.8/dist-packages/gemBS/gemBSbinaries/bs_call' finished with -6
2021-01-20 05:51:18,765 ERROR: Additional threads:  10 7 7
2021-01-20 05:51:18,765 ERROR: Loading reference sequence index
2021-01-20 05:51:18,765 ERROR: Sequence index read in successfully
2021-01-20 05:51:18,765 ERROR: Opened ./mapping/pbat_data/pbat_data.bam for input (Index)
2021-01-20 05:51:18,765 ERROR: Processing region chr3:1-198295559
2021-01-20 05:51:18,765 ERROR: Processing chromosome chr3 (OK)
2021-01-20 05:51:18,765 ERROR: Loading reference for chr3
2021-01-20 05:51:18,765 ERROR: len = 198295559
2021-01-20 05:51:18,766 ERROR: Read in 198285559 bases on chr3 from 10001 - 198295559, stored in 79314226 bytes
2021-01-20 05:51:18,766 ERROR: bs_call: get_template_vector.c:239: read_input: Assertion `al->forward_position == thash->al->forward_position && al->reverse_position == thash->al->reverse_position' failed.
Exception in thread Thread-3:

[tkik]

Hi,
Do you have any updates on this issue?

[heathsc]

It sounds like there may be a problem with the input FASTQ file. Is there a possibility that the read IDs are not unique? If a small dataset showing this problem could be shared then I should be able to track down the cause. Simon

…

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[ifishlin]

@heathsc Hi, I created a small BAM file that will fail in the calling step. please check the email.