Parse error at line

[marcotoffoli]

Good morning,

I'm having an issue with alignment and subsequent sorting of files.
My pipeline is: sequencing with MinION (Nanopore), basecalling and demultiplexing with Guppy, alignment with NGMLR, sorting with samtools. I'm using the latest version of all software.

The command for NGMLR is:
ngmlr -t 8 -r human_hg19_mod.fa -q file.fastq -o output.bam -x ont

With this, I get:
Done (3591 reads mapped (7.32%), 45467 reads not mapped, 49449 lines written)(elapsed: 1m, 51 r/s)

Then I run:
samtools sort output.bam > sorted.bam

and get:
[W::sam_read1] Parse error at line 10440 samtools sort: truncated file. Aborting

So my questions are: why is NGMLR mapping only 7.32% of reads?
What is the problem with samtools sort?

Thank you,
Marco

[marcotoffoli]

Just a quick update: I went back to samtools version 1.9 (i was previously using 1.10)
And the sorting worked without a problem.

Any idea why this is happening?

[wdecoster]

Does ngmlr actually write in BAM format? Is output.bam maybe a SAM file?
Also, I would suggest you to use samtools sort output.bam -o sorted.bam to make sure the output format is bam.

[marcotoffoli]

Hi wdecoster,

From NGMLR help:
-o <string>, --output <string> Adds RG:Z:<string> to all alignments in SAM/BAM

So I'm guessing NGMLR can output a .bam file?

Also, I used the very sam epipeline a lot in the past and didn't get any error. The parse error appeared only after upgrading to samtools 1.10. After going back to samtools 1.9 the error disappeared.

So I'm guessing samtools 1.10 doesn't like the output from NGMLR?

[wdecoster]

That NGMLR help string is actually a mistake, and what you just pasted is the help of --rg-id, but that's something that's wrong in the code and should not be related to this issue.

I don't know if you can write a bam, and perhaps you should just check with the unix file command: file output.bam and see what you get. SAM is text, BAM would return you "gzip compressed data, extra field". Here are the changes to samtools in 1.10: https://github.com/samtools/samtools/releases

[marcotoffoli]

Dear wdecoster,

You are actually right, the output from NGMLR is a SAM file, even though I called it .bam.
I just realigned with NGMLR using .sam for output, but when I then try to run samtools view:
samtools view -b file.sam -o file.bam

I get the error :
[W::sam_read1] Parse error at line 32273 [main_samview] truncated file.

Any idea?

[wdecoster]

I would try to take a look at that line, and see if I can spot what's wrong. Is it the last line of the file?

[marcotoffoli]

If I try to run the same command
samtools view -b file.sam -o file.bam

with samtools 1.9, with the same input file, it works without returning the parse error.

[marcotoffoli]

The offending line is:
a41c1f02-4a89-4bd3-b44b-68b3a71f10d9 2048 1 155188772 -2147483648 8705S14M1I5M1D65M1D6M1I4M2D4M1D29M1I1M1D1M1D1M1I35M1I7M1D1M1D15M48S TACTTGTACTTCATTCAGTTACGTATTGCTGGTGCTGAAAGTTGTCGGTGTCTTTGTGTTAACCTACTTGCCTGTCGCTCTATCTTCCAACCTTTCTTCCTTCTTCTCAATGGGCCCAGGACCTTGACGGCCAGCAGCGGGCTCCAAGGCCCAGGCTTTTACCAACATGACAGGCTACTGGCTGGGCCCAGGCAAGGGGCCTTGGCAGGAAAGTTCCTTGCTGTACCTCCACTGCACTCAGAGGCCAGTGAGGGGGTGCCAAGACAGGACTCCTTCCTCCCCTGGTGAAGTGCCCTTGCAGCTCCCCAGCGTCCACGCCATGGATATTTCCTCCACAGAAGTACAATGCTGATTATGATCTGTCAGCTCAGCAAGGGCAGACACCCTGGCCTTCATGTCTCTCCTGGAGGAGAAGTTGATCCCAGTGCTGGTGAGTGTGCCCAGACCTCCCAGCATCCATGGCCAGCCAGGGAGGGACGGGCACACACAGACCCACAGAGACTCAGGGAGGCATGGAGGTCAGAAGCCCACCTTGAATCAGACAGGTGCACTGGCTCAGACCTGCCTGTTCCTTCCTGCCCACCCAATCCAGGTACATACTTTTTGGATGAGCACCAAGAACTACGTAGAAGTGACCCGGAAGTGGTATGCAGAGGCTATGCCCTTTCCCTCAGCTTCTTCCTGCCTGGCCGCATGCAGCGACAGTGCATGGAACGGCTACAGCTGCTGACTAGAGACTTTGACTGAGATGAGAAGAGTTGGAGAAGGAGGTAGCTCTGAGACCGGGGCTATTGTATGAGATTGAGCCAAGGATGCTGGCCAGGAATGGGAGTGCTTAGAGTGCAGAGGTGGCACATTCACAGCTACCAAAACCTACCTGTGTCGCCCCTACAGCTGTACCAAGAGGCTCAGGAGTGTCTGACCCTGCTCTCTCAGCGCCTGGGCTCTCAAAAAAGTTCTTCTTTGGAGATGCGTGAGTCTGACTCCAGAAGGTGCAATGGGTGGCTTGGAAGAAGATGCAGGTTCAGATGGAGCAGCTGGAGCTGGGGCTGGGGCTGGGGCTGGCTCAGGCTCTGGATAGGAGGTCCCTGAGACAGATACTGGCTGGTGACAGTGGGGCGTGGGGCCAGAGCCTTCTCAGGAGTACAAGGGGTAGGGTGGGAGGGCAGCCAGGCACAGGAAGGGCCTGAAAGAGCTGTGGGGCACTGAGTGTGCCCTTTATGCAGCCCTGGGATGAGCCCTATTCAGGGCCAGGCTGGCAGCACCACCTGGGGATCTCTCCCCATACCAGGTCTAGGACTGTGTGTCCTGTCCTTCCCTAAGTGGCCGCCTGCTGCCCAGAGCCCACCTCCCAAGGCTGACTCTTCCTCCGGCTCCATCTTTACCCCTTCTACCCCAGTGGTTCTCCTCCATCCCACCCTTCTCTCTCTGCTCCAGCCACCTGCCTCCTTGGACACCTTCACGTCTTCAGCTACTTGGCCCTGCTGCTGCAGGCAAAGCTGCCCAGTGGGAGCTGCAAGTCCACCTGCGTGGGCTGCACAACCTCTGTGCCTATTGTACCCACATTCTCAGTCTCTACTTCCCCTGGGATGGAGGTAAGGGGCAGATGGGAGGGGCAGCCCTGGGGGAGTGGGCAGGGATCAAGAACTAGTTCTCCTAACACACCTTCCTTCCTTGACCCTCAGCTGAGGTACCACCGCAGCACTTGAACACCAGCAGGCCCAGAGACTGAGGAGGAATATACCGGCGCCGGAACCAGGTCCTACCTGTGCTGGCAGGTTTGGCAGCCATGGTGGGCTACGCCTTGCTCAGCGGCATTGTCTCCATCCAGCGGGCAACGCCTGCTCCGGGCCCAGGCACCGGACCCTGGGCATGGCTGAGGAGGATGAGAGATGATTTGTCCTCACGCTCCCAAGACTGGTTTTCTACTCTCATGCATTCCAGAGGCCCCCGTGCCTCCTCGTTATTGGTACAGCCGGACGCTGCGGTGCTGCCACCCAGAATAAAGCCACTCACATGACTGGGCTCAAACATTTTCTCCTTTAAGAGCTGCCATTTTCCTGGCTGGTGCCATAGGAATCATCTGGGTGCCTGGGCACACCCGCTGCTGCTTTAAGGCTTCCGCCCTGATGCTGACACTGCTGCTCCACGGGCCCAGTTCTGCATCTCCAGGAAAGACAAACAGTCTCCAGTTTGGGCCCAGCTTTCCTAGTCTCTTCTTTCCTTACCCTCAGCCCTGATCTTGTGTTTGTACAGGACAGTGAGCTCACCCTAGGCCTGGACCCAGGCCCAGTTTCTAAAGCAAGCAGCACACAGCGCATGTTCACATAAGCATGGGGCTGGGGGACACTGGGGCTTACTGATCTTTTTCTAGGCCTCCCAGCCTATGGCACCACCTAGAGGAGAAGTGAGTCACCCAAACCATTGCCCCCTGGGCTTACGTCGCTGTAAGCTCACACTGGCCCTGCTGTGCCCTCTTTAGTCACAGACAGCGTGTGAGCTGACTCTGTCCTTTAATGCCCAGGCTGAGCCCAGTGCCTCCTTGGTATCTGCTCTATCACTGGCGACGCCACAGGCCAGGTGTGAATGGAGTAGCCAGGTGAGATTGTCTCAGGAAGCCCACAGCAGGATCCTTGATGGTAAGAGGCACATCCTTAGAGGCTAGGGAGCAGGGAGGAGAAGCTGAGAGTGTGATCCTGCCAAGGCCCCCAACGCTGTCTTCAGCCCACTTCCCCAGACCTCACCATTGCCCTCACCGGTTTAGCACGACCACAACAGCAGAGCCATCGGGATGCATCAGTGCCACTGCGTCCAGGTCGTTCTTCTGACTGGCAACCAGCCCCACTCTCTGGGAGCCCTCAGGAATGAACTTGCTGAATAACAGAGACAGATGGTCAGAGTCCCCTCAGGGTACCTCCATGAAACCCTCATCTAAGAAGTCACCCACCCTGGACCCACCCCATAACTCCTGCAGAGGCTCTGCCCTGGCTCTCTAGGCCTGGAGCCATGCTGCTGGGCACTGACCCTGCTTTTCTGCATCGCAGTCCAGCCTCAGGCATTGGGGTTTTCTGTTGCTACCTAGTCACTTCCCACTGCCTCCATGGCGCAAAAGGGGATGGGTGTGCTCTTCGAGGTTCCACCTTGAACACCTTCCTGCTCCCTCGTGGTGTAGAGTGATGTAAGCCACCCGATGTGGGGATGATAGGCCTGGTATGGAATGGGGTGCCGCCCTCCACTCACCTGAAGTGGCCAAGGTGGTAGAACATGGGCTGTTTGTAAAACGTGCCCTTGGTGATGTCTACAATGATGGGACTGTCGACAAAAGTTACGCACCCAATTGGGTCCTCCTTCGGGGTTCAGGGCAAGGTTCCAGTCGGTCCAGCCCGACCACATGGTACAGGAGGTTCTGAGGTAAGGACAAGGCAAAGAGACAAGGCTCAACACTGGGGGTCCCCAGAGAGTGTAGGTAAGGGTCACATGTGGGAGAGGCAGCTGTGGGTAGGTCAGCCCTGTGAGGGCACATTCCTTAGTAGCTAAGGAGTTGGGGGTGTGAAGATCCAGGCATCTCAAGGGGAGCTGAAGTCTGAGGCAGCTGCAAGTGCCTCAGTAGTTGCAAAGGGGCAATGAGGTGTGCAGACCTGTGAAGGGAAAGGGAAGATAGGGAATCCATGGTTCCCCAGAGTTGCTCAAAAGGGCAGGCTAGCTGGGAAGCTGGACAGGAAGGGCTTCTGTCAGTCTTTGGTGAAACTAGTAAGAGGTCTGAGGTCTGCTTTGCAGGAAGGGAGACTGGGGTGGCTTACCGTGATGATGCTGTGGCTGTACTGCATCCCTCGATCCCAGGAGCTAGCCGCACACTCTGCTCCCAGAACTTGGAGCCCACACACAGGCCTCTGAGGCAAGAGCATGTGTTGGGGAACAGGCGGTATTGTGCTCCCCTAGGGTGTGGCTTTGGCTGGAGCCAGAAAGTCCAGGTACCAATGTACAGCAATGCCATGAACATATTTAGCTGCTTCTGGGTCTGTCGGTACCTGCAAGGAAGAGCAGCGATCCTGGACCTTGCACACAGGCTTCTGGAACTTCTAGTTCCTGTTGTAGGAATCCTGGAGGTGGGTGACGGGAAGAATGCAGCTAGAGAGGTTTGGGGAGATTTTTTTGTTTTTGAGACAGGATGTGTCACTCTGTTCGGGCTGGAGTACAGTGGCGCAATCACGACTCACTGCAGCCTTGACTTCCTAGGGTCAACTGATCCTCCCACATTAGCCTCCTGAGTAGCTGGGACTACACGGGTGCCACACCCAGCTAATTTGTGTGTGTATGTGTGTGTATGTATGTGTGTGTGTGTATATATATATACATATAAACACATATATATGTATATACACATACAGCCATGAACCACCACCCCCAGCCTAGATAGTTTTGTTTGTTTTGTTTCGAGATGGAGTCTCGCTCAGCCTCCCAGGCTGGAGTGCAGTGGCGCGATCTCGGCTCACCACAGCCACCATCTCCCGGGTTCAGCGGTCTCCCTTCTCAGCCTCCTGAGTAGCTGGGATTACAGACACCCACCATCATGCCAGATAATTTTTTTTTTGTATTTTAGTAGACACAGGGTTTCAACACGTTGGCCAGGGTGGTCTTAAACTCCTGACCTCAGGTGATCACCCGCCTCGGCTTCCCAAAGTGCTGGGTTTGCATGAGTGAGCCACCTCGCCCAGCCCCTAGAAAGGTTTCAAGCGACAACTGTGGGATCCATGGCACCCTGGAGGTCAGGGAATGATGCTCTAGGGAATCCATAGTTGGGTAAGAGAAATCGCTCTAAGTTTGGGAGCCAGTCATTTGGATGCTGGATTTGAAGGTCACTGGAGCACCATGGAGGTCAGGCCTTACCACCTTTGCCCAGTGGGGCAGCAGCAAGCGTTGGTCATCCAGCATGAGTAGGCGGACATTGTGGTGAGAATTTACTGTTGGCGAGGGTAGGACCTAGGTCACGGGCAATGAAGTCTCGCTGATGTTCAGGGGTGAAGCCCAGGCACTGGAAGGAGTATCCACTCAACAGCCCAGCAGAAGGCTCATTCTCAGCTGTACTGCCCAGAACTGTAACTTGTGCTCAGCATAGGCATCCAGGAACCTGGCAAGAGAAAGGTCATGAATGATCCGGCCAAAGAAAGTGGACCAGACCAGCTGGGTGTGGTGGCTCACACCTGTGAATCCCAGCACTTTGGAAGCCGAGGCAGGTGGATCACTTGAGTTCAGGAGTTCGAGAACAGCCTGGCGGAACCCCGTCTCTACTAAAAATAGAAAAATCAGCCGGGCCTGGTGGCAGGCGCCTATAATCCAGCTACTTGGAAGGCTGAGGCAGGAGAATTGCTTGAACTCAGGAGGCAAGGTTACAGTGAGTGAAGATGGCGCCACAGCACTCCAGCCTGGGTGACAGAGAGACTCCTTCTCAAAAAAAAATAAAAAGAAGAAAAATAAAAAAGAAAGTGGGCCAGACCGAGAGAACAGGAAGCCTGATGGAGTGGGCAAGATTGACAGGCCCAAGGCTGAGCCCAGAAGGTAGAAAGGTGGGCTGAGGACAGGCAGATCTGGAAGTGGAACTAGGTTGAGGGTTGGGACACAGATCCAGCATGGCTAAATGGGAGAGCCAGTCCTGATCCACGTCCTTGCTGATCCCTTACTTCACAAAGTATCTGGCCCAGGTCTGGTGGTAGATGTCTCGGGCTGTCCCTTGAGTGACCCCTTCCCATTCACCGCTCCATTGGTCTTGAGCCAAGTGGGTGATGTCCAGGGCTGGCAAGGAGTGAAACGGGACGCTGGGCCAACTGCAGGGCTCAGTGAATCCATCTAGAGACAAAGGTAGTGAAGAGAAGCACCCAGAGTTGGAACACATACTAGCCCAACCAGTGCATCCGGTTCAGCCATTAGCCCCACCCTCCCGCCCCCAGGACAAAACAGCAGGGGACAAAATGTCTGTACAAGCAGACCTACCCTACAGTTTCTCCAACCCCCAGACATCAGGGCCCTCAGGGCCTGAAGCTAAGAATGCCTACCTTGAGCTTGGTATCTTCCTCTGGGAGGCTGAAGTTGTGCAACTGGAAATCATCAGGGGTGTCTGCATAGGTGTAGGTGCGGATGGAGAAGTCACAGCTGGCCATGGGTACCCGGATGATGTTATATCCGATTCCTACAGAAAGGATAGTCAAGACATGGTAGTCCGAGTCAATAGGAGAGTAGGACTCTGCTTATCTTGCCAGTCCCTAATAGTGTCTGAGTCAGGGCCAAGACTGGGCTCCTGGGTTGGAACCTGTGGAGGCTGGCACCTGGGTGGTACCCAGGCCTTTCTGAGCCTGAGTCCGTAGCAGTTAGCAGATGATAGGCAGGGAAATCTTATTTCACAGGGCATTAAAACAGGAACCAAATGTCAGGGATGGGCAGAAGTCGGGGCCCAAAGAAGGGCAAAGAAAAGTGTCAGTGGCTCACGCATGTAATCCCGGCCTTTGGGAGGCCGATGTGGGCAGATCACGAGGTCAGGAGTTCGCAATCATCCTGGCCAACATAGTGAAACCCTGTCTCTACTAAAAATACAAAAATTAACGGGCGTGGTGGCAGGGCACCTGTAATCCCAGGTACTCGAGAGGCTGAGACAGGAGAATCACCAGACGGGAGGTGGAGGTTGCAGTGAGCTGAGATTGCTGCCCACTGCACTCCAGCCTGGGTGACAGTGCGAGACTCTGTCTCAAAAAAAAAAAAGAAAAAGAAAAGGTGTCTGCTGGGCTCGGTGGCTCACACTTATAATTCCAGCACTTCGGGAGGCCAAGGCAGGTATATCATTTGAGGTCAGGAGTTTAAGACTAGCCTGGTCAACATGATGAAACCCTGTCTCTACTAAAAATACAAAAATTGGCAGGTGGTAGTGGCGCACGCCTATAATTCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCACTATAGCCTGGAGGCAGAGGTTGCTGTGAGGGAGATCACACCACTGCACTCCTGTCTCGCCGACAGAATGGGCGGAAGAGTGAGATTCTGCCTCAAAAAAGGCTTAAAAAAGAAAAGAAAAATAGAAAAGTTTCAATGGCTCTATGTCATCTTGTCCCCTTCCTCCTCACCTTCTTCAGAAGTACGATTTAAATGCTGAAATTTTGGGCAGGGGTGACAGGGCAAGGATGTTGAGAACAGCTGCATCTGTCATGGCCCCTCCAAATCCCTTCACTTTCTGGAACTTCTGTTCTGGCTGCAGGGTCAGTATTAGCAGGCCTGAGGACATCCACAGGAATAAGATTATCGTACCCAGCGGGAAGCTCCATGGTGATCACTGACACCATTTACCTCTAGGAGGACCCAGCCTGGCCGGGGGTGAGGGAGTGTAATGGTTACCTGTGCCCGTGTGATTAGCCTGGATGGGCCTCAGCTCATCCGTCGCCCACTGCGTGTACTCTCATAGCGGCTGAAGGTACCAAGGGCAGGGAAGGTCGGGGGTCAAAGGAGTCACAGTATGTGGCATTGCAGACACACACCACCGAGCTGTAGCCGAAGCTTTTAGGGATGCAGGGGCTGGGTACCTGGGAGGAGGGAGTACAAGCAGAGTGAGGTCTGATGAAGACATGGAAATGGACACATCTGCTAGGAGAGACTGAACACGGTTTCAAAATTCCTCACCCTTGGCCGGGCGCAGGGGCTCACACCTGTAATCCTAGCACTTTAGGAGGCCGAGGTGGGCGGATCACCTGAGGTGAGGAGTTTGAGACCTGCCTTGCCAGCATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCTGGGCGTAGTGGTGGCCGCCTGTAATCCCAGCTACCTGGGAAGCTGAAGCAAGAGAATCGCTTGAATCTGGGAGGCAGAGGTTGGAATGAGCCAAATTGCACCACTGCACTCCAGCCCAGGCAACAGAGGCAAGACTCTGTTTCAAAGAAAAAAACAAAATCCTCACCCCAAAGTTGGTCCAGTCACTCAAAAGGATTTATTGAGCACCTACTAAAGTCTACCACCAGCTTACTGGAAGGCTACCAAGGACTATGAGGCAGAAGGAGGCTCTGTGCTACCTCCCCACTGCCTTGACTCACTCACCTGATACTTGGTACACTGCCTAGTAGAAGCAATCCCTGTGAGGCTGCCAGCCTTGTTACCCTACTCAAAGGCTTGGGACATTCCTGAGGACAGAATGAGGAATGACTGAAAAGCAGCCCCTCTCCACCCCTCAACTACTCTCCTGGGCAGGGCTTAGCTGCCTTTGGGTGCCCATATTTGATCTCCCATTCATTAGGACAAGGCCCACAGAAACCTGGGTGCAGCTCTCCCTGCAACCCTTCTGATGACAACTCTCTGCCACCCAAATCAAGGATTGCTCCCCACCACTCTTCCCACCCATTTCAACTAGGCCCCTCCCTCCATCTGTGCCTTGCTCAAAGAGCCATGATGGCCCTGGATTCAAAGAGAGTCTGTCATTCATTAAATTCAGTGCCAGGATTCCAGAAGCACTGTGACAATGCTGATTGGGAGCTCTCTCTCTTACCTCTCTGGAAGGACTTGAAAACTCCATCCCCTCAGGGTCATTAGATGAAGAAGACCACAGGGGTTCCAGAGTCTCTGAAGGATAGAGGATCCACTAAACAAAAACAAGGATGCAGGTACCTGCCTTGGCTAGGCACTAGGTTAGCCCCTCAAGTAATTCCGGCTTCCCCGTGTGGATGGGTCATGTGATGACTAGGAGCGTCACACGACACAGGAGTGAGAGCAACCTGTATATTTCTAAAGGGCAATTGGCTTCCTCTCATCTGTTACAGATTATATGCCCTATAAAACTCTGGAGGCATGTATGGGTGACAACTTTAGGAGCAATATCAGCACCAACAGAAAGGTTAACACAAAGACACCGACAACTT 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AS:i:201 NM:i:29 XI:f:0.8564 XS:i:0 XE:i:201 XR:i:193 MD:Z:15T3^G10T8A0A28A6T8^A10^TT0G1A1^C0C29^G1^G15C0A10T8G1A4^T1^A0A14 SV:i:2 SA:Z:1,155202292,+,86S49M3I18M1D11M1I1M2D26M1D14M1D42M1D9M1I17M1I95M1D99M1D24M2D8M1I4M1D99M1I2M1D55M1D65M2D4M1D2M1I8M1D5M1D32M1D17M1I4M2D33M1I15M1D6M2D4M1I2M1I80M2I36M1D7M2I108M2D16M5D20M1I2M1D4M1D42M1I45M1D25M1I2M2I55M1I118M2I20M2I48M1D116M1D14M1D67M1I29M1D108M1I4M1D8M1D29M1D3M1D1M1D29M1D62M1I33M1D38M1D137M1D27M1D35M1I80M1D6M1D32M2D5M1I26M1D22M1I82M1D35M1D31M1I34M1D50M2D70M1I153M1I15M1I14M1D33M1D139M1I2M2I31M1D5M1D65M1D25M1D6M1D95M1I61M1I25M1I1M1D9M1D13M1D83M1D65M2D34M1D31M1I20M1I38M1D3M1D135M1D40M2I12M1D8M1D19M1I4M1I13M2I87M1D13M1D102M2D25M2I8M3D240M1D97M1D1M1D53M1D8M2D186M1D2M1D15M1I17M1I74M1D78M4I124M1D77M1I44M1I14M1D114M1D50M1D55M4D18M1I15M1I72M2D1M1D75M1I19M1I12M1D62M1D70M1D54M5D27M2D59M1D73M1I32M3D5M1I151M1D7M1I1M1D34M2D14M2D8M1I24M1D2M2D1M1D2M1D41M1I3M1D48M1I2M1D70M1D44M1D90M1D8M1D15M1I50M1D1M2D32M1I2M1I42M1D13M1D10M1I140M1D72M1D20M1D44M3I29M1D17M1I57M2D14M1D2M2I12M1D99M3I21M1D14M1D64M1D2M1D11M1I41M2D2M4D6M1D61M1D81M1I12M1D41M1D49M1D236M1D35M1I15M1D4M1D32M1D33M1D32M1D18M1D46M1D6M1I9M2D12M1I18M1D1M2D2M1D59M1D61M1D13M2D70M1D3M1D8M1I4M1I32M1D81M1D108M2D68M251S,36,395; QS:i:8705 QE:i:8898 CV:f:2.157389

[wdecoster]

That looks like a very long read. Perhaps you should try alignment with the --bam-fix argument

[Laurenz0908]

I have the same problem using ngmlr version 0.2.7 and samtools 1.11
--bam-fix did not help
It occurs on several lines, one is for example:
m54067_210510_000837/8520076/128701_137638 2064 tig00002724 1759331 -2147483648 8840S51M1D61M * 0 0AAAAAGCCGCATACACAGCTTTACGCCGTCAGGTTCCTTCCATTGATATGCAAATTCCAGAAGATATCGCGGTAGAGCAATCCGATTTCTTGCAGGCTCTCAAGGAATCAAACCAGCAGTACTTCGTAGCATGGAAGTTGAAGTTCCTCATGTGGAATGGGAAGATATTGGCGGCTTGGAAACCATAAAACAGACCTTGCGGGAATCTGTGGAAGGGGCTTTGTTGTACCCAGAACTCTACAAAGAAACAAAAGCCAGAGCGCCCAAAGGAATACTGTTGTGGGGTCCTCCCGGTACTGGTAAAACATTATTAGCAAGGCTGTGGCTTCTCAAGCCCGAGCTAACTTTATTGGTATTAATGGACCAGATTTACTCAGCCGTTGGGTGGGAGCCAGTGAACAGGCGGTAAGAGAATTATTTGCTAAAGCCCGACAAGCAGATCCCTGTGTAATATTTATCGATGAACTGGATACATTAGCTCCAGCACGGGGAACTTACACTGGTGATTCTGGGGTAAGTAACAGGGTAGTGGGGCAATTACTGACGGAGTTAGATGGTTTAGAATCGGGGTCTAACATTTTGGTAATTGGGGCTACCAATCGTCCTGATGCCATAGACCCTGCTTTGTTACGTGCAGGACGGTTAGATTTACAATTGAAGGTAGATTTACCAAATTTAGATAGTCGGTTCAAGATTTTACAAGTTTACAATCAGGGCAGACCTCTGTTAAATGTGGACTTGGAACATTGGGCTAAGATAACAGAAGGTTGGAATGGTGCAGATTTAGTATTACTCTGCAATCAAGCCGCTGTAGGAGCAATTCGCCGTTTTAGGTCTCAAGGTGAAACAGATACTGCTGCGATCAAGATTACTGTTGATGATTTCCAAGCTTCTTATGAAGCTTTAAGCCAGCAACGTACAGTTTAGTTCAACCTACGCTTTGGGGCAATTTATAAATTGCCCTAATCCTCTGCTTAAAGAACTGCACAAGCGGCGGGTCGCCCGTACGCCAGAACTCGCTAAGCGCTAAATTGGACGCAATGAGGACAGACCACTAAAACCTGGTACGCAGTAATGTTTCCGTCCCCTGACTGGGAATTGGTAGGGTTGGACTCTATAGGTTTGAATATTTTATCGAAAATTTTGGGTTTAAAACCCCGTCGTTCTACGACGGCTTTTCTTGGTTTTTAATGTAAGACTTCAGAACCTCTAATGGTGCGCCACCGATTGAAGATACAAAATAACTAGGCGACCATAAAGATTCTTTTCCGTAGGGTTTAGGGAATCTGGCTTGCCCATATCTACGACTGTCAACTCCTTTTAAAGCATTTACTATGACAGAAATAGAGAGTTGAGGAGGGTATTCAACTAAGGTATGAATATGGTCTGACTCCCCATTAAATTCCAGCACTTGAAAATTCATTTTTTCAGCCACTTCTCGAAAATACTTCAGGATTAACATCAGGCTCTCCACTGTCAAGATTTGGCGACGATACTTAGTCACACAGACCAAATGAATCTTTAAATCCGAAACAGACGCTCTTTTCGTCAAAGGGTTGACATCTACCAGACTCCTAGCTATAATTAATGCAGACTCATACATTTTTACAGTATGAAGCTAAGATATCGATATCGAATTTACCCAACAGACCAACAATAGAGATTGATGTCTCAATTGTTCGGATGCTGTCGGGTAATATTTAACGATGCCTTAGCATATTGTCAAGAACAATACCGTGCGGGTAACAAAAAGCCTAATATCAACGAACTTTCTCAAAGACTTACAGAACTCAAAAAAACCGAGGAAAAAATCTGGTTGGGAGAAGTTTCTTCTATTCCCTTACAACAGTCTTTAAGAGATTTAGAGCAGGCTTACTCTAACTTTTTCAAATCCTGTAAAGGACAAAGAAAAGGTAAGAAAGTTAAACCTCCCAAGTTTAAAAAGCGTAAATCTAAGCAATCCGCTAGATTTATGGATAATGGTTTTAAACACCTTAATTCCGATTACATTTACATCGCTAAAATTGGTGATATTAAAGTAGTATGGAGTAGAGAGTTACCCACAAAACCTTCTAGTGCTACCTTAATCAAAAACAGTGCTGATAGGTATTTTGTCAGTTTTGTTGTTGAGTTTAATCCTCAACCCTTACCGGAAAACCAAAATTCTGTAGGAATTGATTTAGGAATCACTGATTTTGCCACATTAAGCAATGGTGATAAACTTAAATGTCCTAAGCCTTTAAAGAAACAACTAAAGCGTTTAAGAAGATGACAACGTAATTGGTCGAGGAAACAGAAAGGCAGTCAAAGAAGGGAAGTTGCTAGAAAGAAATTAGCTAAACTTCACGCCAAGATTTCTGATACCAGAAACGATTTTCTTCAGAAATTGTCAACTCGGATTATTCGTGAAAACCAAACGATAGTCTTGGAAGATTTAAAGGTTTCAGGGATGATGAAAAACCGAAAACTATCCCGTGCTATTTCGGATTTGGGTTGGCGCAGTTTGAGAACAATGCTAGAAGTTAAATCTGTTATGTACGGGCGTGATTTTCGTGTCATTGATAGATGGATTCCCACTTCTCAAACCGGTTCTGGCTGCGGTTTTCGCGGTGGCAAAAAAGAATTAAATATGAGATAGTGGACTTGTTTAAATTGTGGCACATCTCATGATAGAGATGTTAACGCTGCGATTAATATTAAGGTCGCCGGAGGGCATTTGGAGACTTTAAACGGATGTGGAGAGAGGGTCAGACTTTCTGTAAAGAAAGCACATCTCAATGAAGCGTCAACCCGTCCAGCATTTCAACAATTGTCAATCTTTGATTTGTTGAAATAGATGGAATCCCCGTTAGCGCACGCCGGGACGGATAGGCGGATGTCAATACAGTCCTCTGTTTCCTAAAGTGCAACTATTTGCAGTTGGGAAGGAAGGCGACAGGCTCACACAGAGGAAACAGCATATGCAGGGCTGACGATCGCTTTAGTGGTGGTAGGTGTCGCGTTTGAGCGAGTCGTAGTTTGGGGTGGGTTGGCTGTGGGAAACGCAGTTGTCTGGTTTTGGATACTTAGGCGATCGCATCCCCGTTTAGTTAGTCGTCTTTTAGAGGCTATTGCCCGGTGGGAAGTGCGGTTGTCTGGTTTTCTAATCTTCGAGAACCCCAAGGCAAGCTTAGTAGGGGCGGGTGCGCGATCAGCCACGGGAAAAACCAGCCAACTATATACACCCGCCCGCGGGTGCGCGATCAGCCATCGGAAACACCAGCACCACTCGCACACAGACCATGCCTTGGCAGCGAAACGCCCAGAGACCCAGCGAACAGAGGAAAAACCAGCCAACCCCCTCAGTACAGGACAAAAAAGCTGAAAGTCTTAGCCAAAAGGGAATCTAGCCGAAAATTCTGAAGGGGGCGCTCCGGGCTGAGGTGAAAGTCGAGGGGTGGGGGGTAGCGCCAAGTTGGGGTGAGAGGGTTAGATCGGAATTACCACAAACCAAACTTCCCTCTCATGAACGAGCTTAACCGATTACGAGACACTTTGCGCCCTCACTTGCCCTGGCACGGGGCGAGATTAAACTTTGTCTGCCTGTTCCTGATGGCGCTATTCCAAACAAAGACGGTTAATCTGATGGAAATAGCGACTGTATTCGCAAATCCTGTGCAAATTTCCTCAAATTACCAGCGATTACAACGTTTTTTTCGGCAATTCAAATTTGACCGGGCAGAGATTGCCCGTTTCGTCGTTAGCCTCATTGACATTCCCCAACCTTGGACTCTTAGTCTCGACCGCACCTGTTGGTCTTTCGGTCAAACCCATTTCAACATCTTGATGTTGGCAGTCGTCCACGAGGGGATTGCCTTTCCCCTGCTGTGGAAGATGCTTGACAAAAAGGGCAATAGCAACAGTGGCGAACGCATGGACTTATTCGACCGCTTCGAGGCACTATTTCCTGACGTGGAGGTGGCTTGTCTGACCGCTGACCGGGAATTTGTGGGGCGAGATTGGCTCTCGTATCTTCTCATCGACCCCCAGGTTCCTTTCCGCCTACGCATCCGCCACAGCGAGCTGATTAGTCCTAAGTTAGGAGGAACTCGGCGTAGCGGCGAACGAATGTTTGATTCTCTGCGACCCGGAGAATTTCGCCAGCTTTCGGGTCGCCGTTGGGTTTGGGGACGGCAGGTTTACGTCATTGGCTCTCGTCTGGCTGATTCGGGGAGTTGTTGATTCTCATCACTAACGCTTGCCCCGAAACGGCCCTCCCCGACTATGCTCGGCGTTGGGGTATTGAAAACCTCTTCGGAGCCTTGAAGACTCGGGGCTTCTGTCTCGAATCGACTCACTTTAAGGACCCTGAGCGCTTGAGCCGTTTATTGGCTTTGCTTAGCCTGGCTTTTATAGCACAAGACAGAACACTTAGGACGTATAATGGAGAGGACGAGATGACTAAGAAGACCAAAAATGCCAGCCCCCTATAGTTACGACCTCAGACAAAAAGTTATTGATGCCATTGAACTAGACGGTATGCCCAAAACAGAAGCCAGTCAAGTTTTCCATGTCAGCCGGAACACCATTAATCTCTGGCTGCAAAGAAAAGCACAGACCGGAGACTTCCTCCCTAAACCTCATCACCGACCTGGCAATAACCACAAAATTACCGACTGGCAAAAATTCAAGGCTTTTGCCCAAGAGCATGGCGACAAAACAGCAGCTCAAATGGCTGAACTTTGGGATGACGACATCTCTCCTCGCACCATATCCAGAGCCTTGAAGAAAATTGGCTTCACCAGAAAAAAAAACTTACGGCTACCAAGAACGTGATGAGCAACAGCGAGAGGAGTTTATGGCTCAGATTGAACAGATGGAGCCACAAGAAGTGGTCTACCTCGATGAAGCCGGCATGAATAGTCAGGACTCGGATTACCCTTATGGTTACTGCGAGGAAGGAAAACGCTTCCATGCACTCAAATCAGGGAAGAGGCAGGGCAGGGTAAGTATGATAGCCGCATGGTGTCATCAACAACTCTTAGCTCCCTTTAGCTTTGAGGGTTGTTGTAATCGGACAGTGTTTGAGTTGTGGTTGGAGTTCATCTTAATTCCAACATTGAAGCCAGGTCAGACTCTAGTATATTGGACAATGCAACGTTTCATAAAGGGGGGCGGATTGCTGAACTGGTGGAGGCAGCTCAATGCCGTTTACTCTATCTTCCGCCTTATTCGCCAGACCTCAACAAGATAGAGAAATGTTGGTCGTGGCTGAAAGCCCGTATTCGCAACTGCACGTGAGCAGTTTGATTCTCTCCATGATGCCATGGATTCCGTTCTCAAAGCTGCGTCCTAACCACCTTGACTAATGCTATACTTGGGCTATGAAGGTGGGTTTGTGGATTCACCAAGGTTCACCCATTCCTTGGAAGGCTCACGGACGACGCTCCCAGAGTCTTTTCCGCACTGGCTTCGATTTTCTACGCCGCACTTTCTCTAATCTGCCTTTGTTTTCAGGGCGGTTTCACCAGGCTCTACAACTTTTGTCCTGTACTTAGGCCAACCCCATACACCAATTATTAATTATTAATTATTAATTATTAATTATTAATTCCAGCCAACTATATACACTCGCCCCCAGGGTGCGCGATCAGCCATCGGAAACACCAGCACCACTCGCACACAGACCATGCCTTGGCAGCGAAACGCCCAGAGACCCAGCGAACAGAGGAAAAACCAGCCAACCCCATACACCAATTATTAATTATTAATTATTAATTATTAATTATTAATTCCAGCCAACTATATACACCCGCCCGCGGGTGCGCGATCAGCCATCGGAAACACCAGCACCACTCGCACACAGACCATGCCTTGGCAGCGAAACGCCCAGAGACCCAGCGAACAGAGGAAAAACCAGCCAACCCCATACACCAATTACAGCCTTTACACAAAACACAAAGTACGGTATAATGTATGGCTATTGTATAAGTTATCACAACCAACTACCATGAGCCGTTACTCTTTAGATTTTCGGAAAAAGATAGTAGAAGCCTATGAAAAAGGAGACACTTCTATCCGAAAAGTAGCGAAGCGCTTTTTGGTGAGTCCAGACACGGTAAGGCGACTGGTCAAACAGTATCGATTAACGGGAGACTTATCTCCTCGTAAGTGTGGCACTAAAAAGAAAAGCATTTGATCTCAGCATGAGGAAGCCGTGATAGATATTGTAGAAGCCCACCCCGACCTGACCTTATGGCAATATAGTGATAAGCTCAGAGACAAGCTGGGCATAAATGTCAGTACGACCATGATAGATAGATTTTTAAAGCAGCACGATATAAGTCTCAAAAAAAACATACAGGAGCGAAAAAGTAGTAACTGAAGAAGTACAGAAAGCGCGAGTAGATTATTGGTAAAGAATTAGAGATGTAGCTCCTGAAAAGCTTGTGTTTATTGATGACAGTGGGTTATGGGTAGGGATGAGCAGACCGTTAGCCAGAGCGACGAAAGGGAAAAAAGTCTATGAACTGCGGAAATCCTATCGAGGTCAAAAAATGACAATAATTGGTGCAATTAAGCTCTCCGGAGTGGTAGCGACTCAAACATTAGAAGGGTCCATGAAAAAGAGGATTTTCTCCAATTCATCAAGTTAGATTTATTGCCAAAATTAAAAAAGGAGATGTAGTGGTAATGGATAACTGAAATTCTCATCATCGAGAAGAAGTCAAAGAAATGATAGAGTCAGTAGGAGCCAGGGTAGAATATCTGCCGGTGTATTCCCTGAGTTTAACCCGATAGAGATGATGTGGTCACAGCTCAAAAGTTTAGTGTGCAAGTTCAGGACGGAGACCATGGAATTATTAGTGAGATTGGTAGAAGTGGCGGTAAGCCTTGTGGATTTACAGTGTTTGAATAACTGGTTTACCAAGTGTTGTTGCTGTGCTTAATGATTGAGATAAAGCCTGTATTAATTATTAATTATTAATTATTAATTATTAATTATTAATTATTAATTCCAGCCAACTATATACACTCGCCCCCAGGGTGCGCGATCAGCCATCGGAAACACCAGCACCACTCGCACACAGACCATGCCTTGGCAGCGAAACGCCCAGAGACCCAGCGAACAGAGGAAAAACCAGCCAACCCCATACACCAATTATTAATTATTAATTATTAATTATTAATTATTAATTCCAGCCAACTATATACACCCGCCCGCGGGTGCGCGATCAGCCATCGGAAACACCAGCACCACTCGCACACAGACCATGCATTGGCAGCGAAACGCCCAGAGACCCAGCGAACAGAGGAAAAACCAGCCAACCCCATACACCAATTATTAATTATTAATTATTAATTATTAATTCCAGCCAACTATATACACCCGCCCCATAGGACCCAAACACCAGCACCACTCGCACACAGCTCTGCCCTTTGCACGGTACGGCTACAAGACTGGTAGAAGAGGGGCGGGTGGTAGTAGGGGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCGCAGGTAGGCTTTGAAAACCCGCCCTTGCGGGTTCCGCAGTCAGCCATAATTCCCACAAATCAACTTTAAAAACCCACCGACGACGATGCGCCTAAGACCATGCACCCCACAAACAGCACAGAACACGCGCCAC * AS:i:219 NM:i:1 XI:f:0.9912 XS:i:0 XE:i:219 XR:i:112 MD:Z:51^C61 SV:i:2 SA:Z:tig00002724,1750485,-,105M1D209M1D2741M1D383M1D770M1D915M2I1477M1D49M1D109M1D2080M112S,22,10; QS:i:8840 QE:i:8952 CV:f:1.251117

[fritzsedlazeck]

@Laurenz0908 just to check is this HiFi data ?

[Laurenz0908]

@fritzsedlazeck yess, and as reported in #83 the read has a negative MAPQ value so I think it's the same problem

[fritzsedlazeck]

Mhm I need to check if that has todo with some samtools update.. thanks for reporting.
Fritz

[pi3rrr3]

Hi, if useful, I face the same issue using ngmlr 0.2.7 and samtools 1.10, with standard PacBio data and two distinct libraries.
Faulty lines also show negative MAPQ values:
m54214_200325_095631/4260092/119840_123419 2064 Scaffold04 8785531 -2147483648 ...
m54217_200324_032638/4260231/60987_76267 2064 Scaffold14 4429481 -2147483648 ...

As mentioned earlier by @marcotoffoli, using samtools 1.9 solves the issue.

[Euphrasiologist]

Can verify also that downgrading samtools also worked for me...

[splaisan]

Hi!
I have today the same error with or without --bam-fix, --rg-id and --rg-sm all set.
Is there an update and fix to have ngmlr work with the current samtools?

ngmlr 0.2.7
samtools 1.15

[skr3178]

I face similar issue when running this tutorial
https://github.com/fenderglass/jax-meta-tutorial
minimap2 -ax map-hifi assembly_graph.fasta SRR13128014_1gb.fastq.gz -t 30 | samtools sort > graph_alignment.bam samtools index graph_alignment.bam

[splaisan]

I face similar issue when running this tutorial https://github.com/fenderglass/jax-meta-tutorial minimap2 -ax map-hifi assembly_graph.fasta SRR13128014_1gb.fastq.gz -t 30 | samtools sort > graph_alignment.bam samtools index graph_alignment.bam

Hi @skr3178, this is a recent recurring issue with samtools installed from bioconda and conflicting with other libraries (ncurses, openssl, ...). A fix is to use a non-conda copy of samtools installed on your server (works here for me)

[skr3178]

Thank you @splaisan .
I used a different reference file and it seemed to work. I didn't try the solution you posted but that may also work.