Failure to parse cell barcodes from bam files

[kevingmonahan]

scTE fails to find cell barcode information in bam files I generated using the cell ranger pipeline:

$ scTE -i possorted_genome_bam.bam -o out_rep2 -x /software/scTE/mm10.exclusive.idx --hdf5 True -CB CB -UMI UB
  DEBUG   : Creating converter from 7 to 5
  DEBUG   : Creating converter from 5 to 7
  DEBUG   : Creating converter from 7 to 5
  DEBUG   : Creating converter from 5 to 7
  INFO    : Parameter list:
  Sample = out_rep2
  Reference annotation index = /software/scTE/mm10.exclusive.idx
  Minimum number of genes required = 200
  Minimum number of counts required = None
  Number of threads = 1
  
  INFO    : Loading the genome annotation index... 2021-04-02 18:14:28
  INFO    : Loaded '/software/scTE/mm10.exclusive.idx' binary file with 3900779 items
  ['1', '10', '11', '12', '13', '14', '15', '16', '17', '18', '19', '2', '3', '4', '5', '6', '7', '8', '9', 'M', 'X', 'Y']
  INFO    : Finished loading the genome annotation index... 2021-04-02 18:15:01
  
  INFO    : Processing BAM/SAM files ...2021-04-02 18:15:01
  ERROR   : The input file possorted_genome_bam.bam has no cell barcodes information, plese make sure the aligner have add the cell barcode key, or set CB to False

The bam files have CB and UB flags:

$ samtools view possorted_genome_bam.bam | head
	A00521:52:HHVH7DMXX:1:2126:5737:27273   16      chr1    3000239 255     91M     *       0       0       TTTCATCCAGGTTTTCCTGGTTTTTTTTTAGTATAGCCTTTCATAGTAGAATCTGATGATGTTTTTGATATCCTCATGTTCTGTTGTTATG     FFFF:FFFFFFFF:FFFFFFFFFFFFF,FFFFF,FFFFF:FFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF      NH:i:1  HI:i:1  AS:i:85 nM:i:2  RG:Z:Rachel_control_rep2:0:1:HHVH7DMXX:1        RE:A:I  xf:i:0  CR:Z:ATTATCCCAGTATGCT   CY:Z:FFFFFFFFFFFFFFFF   CB:Z:ATTATCCCAGTATGCT-1 UR:Z:AGGTCCACTT UY:Z:FFFFFFFFFF UB:Z:AGGTCCACTT
	A00521:52:HHVH7DMXX:1:2126:5936:27398   16      chr1    3000239 255     91M     *       0       0       TTTCATCCAGGTTTTCCTGGTTTTTTTTTAGTATAGCCTTTCATAGTAGAATCTGATGATGTTTTTGATATCCTCATGTTCTGTTGTTATG     FF:FFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF      NH:i:1  HI:i:1  AS:i:85 nM:i:2  RG:Z:Rachel_control_rep2:0:1:HHVH7DMXX:1        RE:A:I  xf:i:0  CR:Z:ATTATCCCAGTATGCT   CY:Z:FFFFFFFFFFFFFFFF   CB:Z:ATTATCCCAGTATGCT-1 UR:Z:AGGTCCACTT UY:Z:FFFFFFFFFF UB:Z:AGGTCCACTT
	A00521:52:HHVH7DMXX:1:1470:6668:19617   16      chr1    3000373 255     91M     *       0       0       TATGCCCTCTAGTTAGTCTGGCTAAGGGTTTATCTATCTTGTTGACTTTCTCAAAGAACCAGCTACTAGTTTGGTTGATTCTTTGAATATT     FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF      NH:i:1  HI:i:1  AS:i:87 nM:i:1  RG:Z:Rachel_control_rep2:0:1:HHVH7DMXX:1        RE:A:I  xf:i:0  CR:Z:ACTTACTCACAGGCCT   CY:Z:FFFFFFFFFFFFFFFF   CB:Z:ACTTACTCACAGGCCT-1 UR:Z:TGGTGTTGGT UY:Z:FFFFFFFFFF UB:Z:TGGTGTTGGT
	A00521:52:HHVH7DMXX:2:1410:7952:4460    16      chr1    3009349 1       1S65M25S        *       0       0       GTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTGAACTAACCCATGTACTCTGCGTTGATACCAC     FFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF      NH:i:3  HI:i:1  AS:i:59 nM:i:2  ts:i:25 RG:Z:Rachel_control_rep2:0:1:HHVH7DMXX:2        RE:A:I  xf:i:0  CR:Z:TAGACCATCCGATATG   CY:Z:FFF:FFFFFFFFFFFF   CB:Z:TAGACCATCCGATATG-1 UR:Z:AACTATAACG UY:Z:FFFFFFFFFF UB:Z:AACTATAACG

[jphe]

Can you check if the bam file has empty CB reads? like this: jphe/scTE#7 (comment)

As if you set -CB CB, while some reads has no CB:Z tag, scTE will report such warnning.

[kevingmonahan]

Yes, there were reads without CB:Z tags. After removing those reads, everything ran fine. Thanks!

[lnrlz]

Yes, there were reads without CB:Z tags. After removing those reads, everything ran fine. Thanks!

Hi kevingmonahan, may I ask how do you filter the reads without barcodes? Thanks a lot!

[kevingmonahan]

I filtered out those lines using samtools and awk.

For example:

samtools view possorted_genome_bam.bam -h | awk '/^@/ || /CB:/' | samtools view -h -b > possorted_genome_bam.clean.bam

[lnrlz]

I filtered out those lines using samtools and awk.

For example:

samtools view possorted_genome_bam.bam -h | awk '/^@/ || /CB:/' | samtools view -h -b > possorted_genome_bam.clean.bam

I appreciate it very much! I tried the same command, but got an error (please see below):
samtools view possorted_genome_bam.bam -h | awk '/^@/ || /CB:/' |samtools view -h -b > possorted_genome_CB_clean.bam

[E::hts_hopen] Failed to open file possorted_genome_bam.bam [E::hts_open_format] Failed to open file possorted_genome_bam.bam samtools view: failed to open "possorted_genome_bam.bam" for reading: Exec format error

The samtools cannot open the bam file, which was produced from 10X Cellranger cloud analysis.
Do you have any idea about the reason for this?
Thanks again!

[kevingmonahan]

it's just not finding your bam file - check your file name

…

On Mon, Jan 30, 2023 at 1:24 PM lnrlz ***@***.***> wrote: I filtered out those lines using samtools and awk. For example: samtools view possorted_genome_bam.bam -h | awk '/^@/ || /CB:/' | samtools view -h -b > possorted_genome_bam.clean.bam I appreciate it very much! I tried the same command, but got an error (please see below): samtools view possorted_genome_bam.bam -h | awk '/^@/ || /CB:/' |samtools view -h -b > possorted_genome_CB_clean.bam [E::hts_hopen] Failed to open file possorted_genome_bam.bam [E::hts_open_format] Failed to open file possorted_genome_bam.bam samtools view: failed to open "possorted_genome_bam.bam" for reading: Exec format error The samtools cannot open the bam file, which was produced from 10X Cellranger cloud analysis. Do you have any idea about the reason for this? Thanks again! — Reply to this email directly, view it on GitHub <#5 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ACHAHDLL6FD4KZJOLWNURRTWVABOJANCNFSM42JQJQ2A> . You are receiving this because you authored the thread.Message ID: ***@***.***>