Need help with Velocyto Analysis. Errors: MemoryError: bam file #0 could not be sorted by cells.

Question

[Anna]

Good afternoon community! I am new in the field and need you help with an ongoing issue I have with velocity analysis. I am running a velocyto analysis on HPC cluster, and using command run. This is the full script I am using:*

module load Anaconda3
source activate tutorial
export LC_ALL=en_US.utf-8
export LANG=en_US.utf-8
velocyto run -vvv \
--bcfile /home/agera4/RNAvelocityHCI003GM/outs/filtered_feature_bc_matrix/barcodes.tsv.gz \
--outputfolder /home/agera4/RNAvelocityHCI003GM/outs \
--samtools-threads 18 \
--samtools-memory 8000 \
/home/agera4/RNAvelocityHCI003GM/outs/possorted_genome_bam.bam \
/home/agera4/genes.gtf

I have downloaded my gif file from the 10xgenomics portal. Below is the error message I am getting. I attempted to manually sort the bam file using samtools sort -b command, but it would never return anything. Did anyone have the same issue? What could it be related to?

2023-04-04 20:37:47,373 - DEBUG - Marking up chromosome KI270589.1
2023-04-04 20:37:47,373 - WARNING - The .bam file refers to a chromosome 'KI270589.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,373 - WARNING - The .bam file refers to a chromosome 'KI270589.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,383 - DEBUG - Marking up chromosome KI270726.1
2023-04-04 20:37:47,383 - WARNING - The .bam file refers to a chromosome 'KI270726.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,383 - WARNING - The .bam file refers to a chromosome 'KI270726.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,383 - DEBUG - Marking up chromosome KI270735.1
2023-04-04 20:37:47,383 - WARNING - The .bam file refers to a chromosome 'KI270735.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,383 - WARNING - The .bam file refers to a chromosome 'KI270735.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,384 - DEBUG - Marking up chromosome KI270711.1
2023-04-04 20:37:47,384 - WARNING - The .bam file refers to a chromosome 'KI270711.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,384 - WARNING - The .bam file refers to a chromosome 'KI270711.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,391 - DEBUG - Marking up chromosome KI270745.1
2023-04-04 20:37:47,391 - WARNING - The .bam file refers to a chromosome 'KI270745.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,391 - WARNING - The .bam file refers to a chromosome 'KI270745.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,402 - DEBUG - Marking up chromosome KI270714.1
2023-04-04 20:37:47,402 - WARNING - The .bam file refers to a chromosome 'KI270714.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,402 - WARNING - The .bam file refers to a chromosome 'KI270714.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,403 - DEBUG - Marking up chromosome KI270732.1
2023-04-04 20:37:47,403 - WARNING - The .bam file refers to a chromosome 'KI270732.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,403 - WARNING - The .bam file refers to a chromosome 'KI270732.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,403 - DEBUG - Marking up chromosome KI270713.1
2023-04-04 20:37:47,403 - WARNING - The .bam file refers to a chromosome 'KI270713.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,403 - WARNING - The .bam file refers to a chromosome 'KI270713.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,406 - DEBUG - Marking up chromosome KI270754.1
2023-04-04 20:37:47,406 - WARNING - The .bam file refers to a chromosome 'KI270754.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,406 - WARNING - The .bam file refers to a chromosome 'KI270754.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,419 - DEBUG - Marking up chromosome KI270710.1
2023-04-04 20:37:47,419 - WARNING - The .bam file refers to a chromosome 'KI270710.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,419 - WARNING - The .bam file refers to a chromosome 'KI270710.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,421 - DEBUG - Marking up chromosome KI270717.1
2023-04-04 20:37:47,421 - WARNING - The .bam file refers to a chromosome 'KI270717.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,421 - WARNING - The .bam file refers to a chromosome 'KI270717.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,421 - DEBUG - Marking up chromosome KI270720.1
2023-04-04 20:37:47,421 - WARNING - The .bam file refers to a chromosome 'KI270720.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,421 - WARNING - The .bam file refers to a chromosome 'KI270720.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,472 - DEBUG - Marking up chromosome KI270718.1
2023-04-04 20:37:47,472 - WARNING - The .bam file refers to a chromosome 'KI270718.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,472 - WARNING - The .bam file refers to a chromosome 'KI270718.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,486 - DEBUG - Marking up chromosome KI270755.1
2023-04-04 20:37:47,486 - WARNING - The .bam file refers to a chromosome 'KI270755.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,486 - WARNING - The .bam file refers to a chromosome 'KI270755.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,486 - DEBUG - Marking up chromosome KI270707.1
2023-04-04 20:37:47,486 - WARNING - The .bam file refers to a chromosome 'KI270707.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,486 - WARNING - The .bam file refers to a chromosome 'KI270707.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,487 - DEBUG - Marking up chromosome KI270366.1
2023-04-04 20:37:47,487 - WARNING - The .bam file refers to a chromosome 'KI270366.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,487 - WARNING - The .bam file refers to a chromosome 'KI270366.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,487 - DEBUG - Marking up chromosome KI270467.1
2023-04-04 20:37:47,487 - WARNING - The .bam file refers to a chromosome 'KI270467.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,487 - WARNING - The .bam file refers to a chromosome 'KI270467.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,659 - DEBUG - Marking up chromosome KI270528.1
2023-04-04 20:37:47,659 - WARNING - The .bam file refers to a chromosome 'KI270528.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,659 - WARNING - The .bam file refers to a chromosome 'KI270528.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,659 - DEBUG - Marking up chromosome KI270333.1
2023-04-04 20:37:47,659 - WARNING - The .bam file refers to a chromosome 'KI270333.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,659 - WARNING - The .bam file refers to a chromosome 'KI270333.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,660 - DEBUG - Marking up chromosome KI270330.1
2023-04-04 20:37:47,660 - WARNING - The .bam file refers to a chromosome 'KI270330.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,660 - WARNING - The .bam file refers to a chromosome 'KI270330.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,661 - DEBUG - Marking up chromosome KI270466.1
2023-04-04 20:37:47,661 - WARNING - The .bam file refers to a chromosome 'KI270466.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,662 - WARNING - The .bam file refers to a chromosome 'KI270466.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,743 - DEBUG - Marking up chromosome KI270337.1
2023-04-04 20:37:47,743 - WARNING - The .bam file refers to a chromosome 'KI270337.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,743 - WARNING - The .bam file refers to a chromosome 'KI270337.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:47,744 - DEBUG - Marking up chromosome KI270336.1
2023-04-04 20:37:47,744 - WARNING - The .bam file refers to a chromosome 'KI270336.1+' not present in the annotation (.gtf) file
2023-04-04 20:37:47,744 - WARNING - The .bam file refers to a chromosome 'KI270336.1-' not present in the annotation (.gtf) file
2023-04-04 20:37:52,626 - DEBUG - Read first 170 million reads
2023-04-04 20:38:03,141 - DEBUG - End of file. Reset index: start scanning from initial position.
2023-04-04 20:38:03,141 - DEBUG - 2901308 reads were skipped because no apropiate cell or umi barcode was found
2023-04-04 20:38:03,142 - INFO - Now just waiting that the bam sorting process terminates
Traceback (most recent call last):
  File "/home/agera4/.conda/envs/tutorial/bin/velocyto", line 11, in <module>
    sys.exit(cli())
  File "/home/agera4/.conda/envs/tutorial/lib/python3.6/site-packages/click/core.py", line 1128, in __call__
    return self.main(*args, **kwargs)
  File "/home/agera4/.conda/envs/tutorial/lib/python3.6/site-packages/click/core.py", line 1053, in main
    rv = self.invoke(ctx)
  File "/home/agera4/.conda/envs/tutorial/lib/python3.6/site-packages/click/core.py", line 1659, in invoke
    return _process_result(sub_ctx.command.invoke(sub_ctx))
  File "/home/agera4/.conda/envs/tutorial/lib/python3.6/site-packages/click/core.py", line 1395, in invoke
    return ctx.invoke(self.callback, **ctx.params)
  File "/home/agera4/.conda/envs/tutorial/lib/python3.6/site-packages/click/core.py", line 754, in invoke
    return __callback(*args, **kwargs)
  File "/home/agera4/.conda/envs/tutorial/lib/python3.6/site-packages/velocyto/commands/run.py", line 116, in run
    samtools_memory=samtools_memory, dump=dump, loom_numeric_dtype=dtype, verbose=verbose, additional_ca=additional_ca)
  File "/home/agera4/.conda/envs/tutorial/lib/python3.6/site-packages/velocyto/commands/_run.py", line 225, in _run
    Otherwise sort manually by samtools ``sort -l [compression] -m [mb_to_use]M -t [tagname] -O BAM -@ [threads_to_use] -o cellsorted_[bamfile] [bamfile]``")
MemoryError: bam file #0 could not be sorted by cells.
                This is probably related to an old version of samtools, please install samtools >= 1.6.                In alternative this could be a memory error, try to set the --samtools_memory option to a value compatible with your system.                 Otherwise sort manually by samtools ``sort -l [compression] -m [mb_to_use]M -t [tagname] -O BAM -@ [threads_to_use] -o cellsorted_[bamfile] [bamfile]``

[rpolicastro]

Have you tried the suggestions the error message provided?

MemoryError: bam file #0 could not be sorted by cells. This is probably related to an old version of samtools, please install samtools >= 1.6. In alternative this could be a memory error, try to set the --samtools_memory option to a value compatible with your system

As a related suggestions velocyto is dated and takes a long time to run. If you can it's better to go through STARsolo, alevin-fry, or kallisto bustools to get your spliced/unspliced counts.

[Anna]

So, yesterday I tried to install samtools 1.11 using conda forge command and indeed, it worked! My only concern that it gave me this message prior to generating loom file:

2023-04-06 01:58:23,765 - DEBUG - Counting for batch 53, containing 100 cells and 2113146 reads
2023-04-06 01:59:13,998 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 01:59:47,372 - DEBUG - Counting for batch 54, containing 100 cells and 2067713 reads
2023-04-06 02:00:34,551 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:01:10,871 - DEBUG - Counting for batch 55, containing 100 cells and 2198696 reads
2023-04-06 02:02:02,179 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:02:11,055 - DEBUG - Read first 140 million reads
2023-04-06 02:02:36,163 - DEBUG - Counting for batch 56, containing 100 cells and 2122480 reads
2023-04-06 02:03:24,093 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:03:58,168 - DEBUG - Counting for batch 57, containing 100 cells and 2038967 reads
2023-04-06 02:04:44,790 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:05:14,377 - DEBUG - Counting for batch 58, containing 100 cells and 1815443 reads
2023-04-06 02:05:56,005 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:06:29,146 - DEBUG - Counting for batch 59, containing 100 cells and 1983653 reads
2023-04-06 02:07:15,678 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:07:29,802 - DEBUG - Read first 150 million reads
2023-04-06 02:07:49,450 - DEBUG - Counting for batch 60, containing 100 cells and 1987572 reads
2023-04-06 02:08:34,875 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:09:08,640 - DEBUG - Counting for batch 61, containing 100 cells and 1984640 reads
2023-04-06 02:09:53,723 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:10:23,488 - DEBUG - Counting for batch 62, containing 100 cells and 1759512 reads
2023-04-06 02:11:03,700 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:11:35,719 - DEBUG - Counting for batch 63, containing 100 cells and 1949645 reads
2023-04-06 02:12:22,034 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:12:39,527 - DEBUG - Read first 160 million reads
2023-04-06 02:12:51,977 - DEBUG - Counting for batch 64, containing 100 cells and 1832995 reads
2023-04-06 02:13:34,045 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:14:10,314 - DEBUG - Counting for batch 65, containing 100 cells and 2332438 reads
2023-04-06 02:15:04,930 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:15:37,596 - DEBUG - Counting for batch 66, containing 100 cells and 2105230 reads
2023-04-06 02:16:26,975 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:16:59,855 - DEBUG - Counting for batch 67, containing 100 cells and 2045972 reads
2023-04-06 02:17:46,628 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:18:05,560 - DEBUG - Read first 170 million reads
2023-04-06 02:18:19,976 - DEBUG - Counting for batch 68, containing 100 cells and 1927368 reads
2023-04-06 02:19:04,940 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:19:33,139 - DEBUG - Counting for batch 69, containing 78 cells and 1720895 reads
2023-04-06 02:20:12,860 - DEBUG - 0 reads not considered because fully enclosed in repeat masked regions
2023-04-06 02:20:18,265 - DEBUG - 2901308 reads were skipped because no apropiate cell or umi barcode was found
2023-04-06 02:20:18,266 - DEBUG - Counting done!
2023-04-06 02:20:18,278 - DEBUG - Generating output file /home/agera4/RNAvelocityHCI003GM/outs/possorted_genome_bam_CE8EQ.loom
2023-04-06 02:20:18,278 - DEBUG - Collecting row attributes
2023-04-06 02:20:18,364 - DEBUG - Generating data table
2023-04-06 02:20:20,515 - DEBUG - Writing loom file
2023-04-06 02:20:34,314 - DEBUG - Terminated Succesfully!
Epilog:  nodes=1:ppn=1

[Ram]

You butchered the code segment that you pasted by formatting it as quoted text instead of using the code option. I've tried to clean it up as best as I can but it could still be wrong. If you can access the original error message, please edit your post and copy-paste it again, this time using the proper 101010 button and NOT THE double quote button.

[Anna]

Thank you for letting me know how to do it!

Answer 1

[ATpoint]

What is your samtools version? Also, samtools total memory consumption is samtools-memory * samtools-threads which would be 8GB * 18 which is 144GB -- does the machine/node has that much? Try to reduce it, you might simply run out of memory causing this crash. Even on a proper HPC node I use "only" 8 cores and 1000 (1GB) memory, beyond that speed gains are limited and do not merit the extensive resources.

[Anna]

yes, you were right, I did this and it generated a loom file:

--samtools-threads 8 \
--samtools-memory 4000 \