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mpileup returns different reference sequence for different bam given same input bed and fasta #2018

Description

@joegeorgeson

Hi samtools,

I'm trying to figure out why samtools mpileup gives different reference sequences for different bam files given that the bam were generated from alignment to the same reference genome (via STAR) and during mpileup I pass the same input bed and fasta.

I initially thought the issue stemmed from another program, but after troubleshooting see that it comes from samtools. Please see my comment on other github, which also contains example data; y9c/cpup#1 (comment)

And of course, if this is user error, please advise!

(run samtools --version)

$ samtools --version
samtools 1.18
Using htslib 1.18
Copyright (C) 2023 Genome Research Ltd.

Samtools compilation details:
    Features:       build=configure curses=yes
    CC:             gcc
    CPPFLAGS:       -I/apps/easybd/easybuild/software/cURL/8.0.1-GCCcore-12.3.0/include -I/apps/easybd/easybuild/software/XZ/5.4.2-GCCcore-12.3.0/include -I/apps/easybd/easybuild/software/bzip2/1.0.8-GCCcore-12.3.0/include -I/apps/easybd/easybuild/software/zlib/1.2.13-GCCcore-12.3.0/include -I/apps/easybd/easybuild/software/ncurses/6.4-GCCcore-12.3.0/include
    CFLAGS:         -O2 -ftree-vectorize -march=x86-64 -mtune=generic -fno-math-errno -fPIC
    LDFLAGS:        -L/apps/easybd/easybuild/software/cURL/8.0.1-GCCcore-12.3.0/lib64 -L/apps/easybd/easybuild/software/cURL/8.0.1-GCCcore-12.3.0/lib -L/apps/easybd/easybuild/software/XZ/5.4.2-GCCcore-12.3.0/lib64 -L/apps/easybd/easybuild/software/XZ/5.4.2-GCCcore-12.3.0/lib -L/apps/easybd/easybuild/software/bzip2/1.0.8-GCCcore-12.3.0/lib64 -L/apps/easybd/easybuild/software/bzip2/1.0.8-GCCcore-12.3.0/lib -L/apps/easybd/easybuild/software/zlib/1.2.13-GCCcore-12.3.0/lib64 -L/apps/easybd/easybuild/software/zlib/1.2.13-GCCcore-12.3.0/lib -L/apps/easybd/easybuild/software/ncurses/6.4-GCCcore-12.3.0/lib64 -L/apps/easybd/easybuild/software/ncurses/6.4-GCCcore-12.3.0/lib -L/apps/easybd/easybuild/software/GCCcore/12.3.0/lib64 -L/apps/easybd/easybuild/software/GCCcore/12.3.0/lib
    HTSDIR:         htslib-1.18
    LIBS:           -lm -lpthread
    CURSES_LIB:     -lncursesw

HTSlib compilation details:
    Features:       build=configure libcurl=yes S3=yes GCS=yes libdeflate=no lzma=yes bzip2=yes plugins=no htscodecs=1.5.1
    CC:             gcc
    CPPFLAGS:       -I/apps/easybd/easybuild/software/cURL/8.0.1-GCCcore-12.3.0/include -I/apps/easybd/easybuild/software/XZ/5.4.2-GCCcore-12.3.0/include -I/apps/easybd/easybuild/software/bzip2/1.0.8-GCCcore-12.3.0/include -I/apps/easybd/easybuild/software/zlib/1.2.13-GCCcore-12.3.0/include -I/apps/easybd/easybuild/software/ncurses/6.4-GCCcore-12.3.0/include
    CFLAGS:         -O2 -ftree-vectorize -march=x86-64 -mtune=generic -fno-math-errno -fPIC
    LDFLAGS:        -L/apps/easybd/easybuild/software/cURL/8.0.1-GCCcore-12.3.0/lib64 -L/apps/easybd/easybuild/software/cURL/8.0.1-GCCcore-12.3.0/lib -L/apps/easybd/easybuild/software/XZ/5.4.2-GCCcore-12.3.0/lib64 -L/apps/easybd/easybuild/software/XZ/5.4.2-GCCcore-12.3.0/lib -L/apps/easybd/easybuild/software/bzip2/1.0.8-GCCcore-12.3.0/lib64 -L/apps/easybd/easybuild/software/bzip2/1.0.8-GCCcore-12.3.0/lib -L/apps/easybd/easybuild/software/zlib/1.2.13-GCCcore-12.3.0/lib64 -L/apps/easybd/easybuild/software/zlib/1.2.13-GCCcore-12.3.0/lib -L/apps/easybd/easybuild/software/ncurses/6.4-GCCcore-12.3.0/lib64 -L/apps/easybd/easybuild/software/ncurses/6.4-GCCcore-12.3.0/lib -L/apps/easybd/easybuild/software/GCCcore/12.3.0/lib64 -L/apps/easybd/easybuild/software/GCCcore/12.3.0/lib -fvisibility=hidden

HTSlib URL scheme handlers present:
    built-in:	 preload, data, file
    S3 Multipart Upload:	 s3w, s3w+https, s3w+http
    Amazon S3:	 s3+https, s3+http, s3
    Google Cloud Storage:	 gs+http, gs+https, gs
    libcurl:	 imaps, pop3, gophers, http, smb, gopher, ftps, imap, smtp, smtps, rtsp, ftp, telnet, mqtt, https, smbs, tftp, pop3s, dict
    crypt4gh-needed:	 crypt4gh
    mem:	 mem

Please describe your environment.

  • OS (run uname -sr on Linux/Mac OS or wmic os get Caption, Version on Windows)
$ uname -sr
Linux 3.10.0-1160.81.1.el7.x86_64
  • machine architecture (run uname -m on Linux/Mac OS or wmic os get OSArchitecture on Windows)
$ uname -m
x86_64
  • compiler (run gcc --version or clang --version)
$ gcc --version
gcc (GCC) 12.3.0
Copyright (C) 2022 Free Software Foundation, Inc.
This is free software; see the source for copying conditions.  There is NO
warranty; not even for MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.

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