Invalid Data

[BrendaLee1]

Hi,
Thank you for this tool, I tried the pipline you provided on my data, but during denovo detection, I got a lot of warning:
2024-11-24 20:55:02 [WARN] - Skipping invalid data in M
2024-11-24 20:55:02 [WARN] - Skipping invalid data in C
2024-11-24 20:55:02 [WARN] - Skipping invalid data in F

the files in input path named as follows:
Sample.spanning.bam
Sample.vcf.gz
Sample.sorted.vcf.gz
Sample.sorted.vcf.gz.csi
Sample.spanning.sorted.bam
Sample.spanning.sorted.bam.bai

but the de-novo output seems very strange:
trid genotype denovo_coverage allele_coverage allele_ratio child_coverage child_ratio mean_diff_father mean_diff_mother father_dropout_prob mother_dropout_prob allele_origin denovo_status per_allele_reads_father per_allele_reads_mother per_allele_reads_child father_dropout mother_dropout child_dropout index father_MC mother_MC child_MC father_AL mother_AL child_AL father_overlap_coverage mother_overlap_coverage
chr19_47047398_47047538_CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC 0 0 0 0.0 0 0.0 0.0 0.0 0.0 0.0 . . . . . . . . 0 . . . . . . . .
chr19_2041258_2041346_GGCCCCAACCA 0 0 0 0.0 0 0.0 0.0 0.0 0.0 0.0 . . . . . . . . 0 . . . . . . . .

All the values were set to zero through the whole output file.

I checked the vcf files of trio data generated by trgt, an exmple showed below:

Father: chr19 47047398 . CAGCCTCGCCCTTCTTTTCCTTCAAATGCCGCCATCTCCTACCGAGTATGGCCTGGGCCAATCCCATCCATGTCCTACCGAGTATGGCCTGGGCCAATCCCACCCACGTCCGTCCCCATTCACGTCCTTTACAAACAGCCC . 0 . TRID=chr19_47047398_47047538_CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC;END=47047538;MOTIFS=CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC;STRUC=(CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC)n GT:AL:ALLR:SD:MC:MS:AP:AM 0/0:140,140:138-142,140-140:20,20:2,2:0(30-102),0(30-102):0.5,0.5:.,.

Mother:
chr19 47047398 . CAGCCTCGCCCTTCTTTTCCTTCAAATGCCGCCATCTCCTACCGAGTATGGCCTGGGCCAATCCCATCCATGTCCTACCGAGTATGGCCTGGGCCAATCCCACCCACGTCCGTCCCCATTCACGTCCTTTACAAACAGCCC . 0 . TRID=chr19_47047398_47047538_CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC;END=47047538;MOTIFS=CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC;STRUC=(CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC)n GT:AL:ALLR:SD:MC:MS:AP:AM 0/0:140,140:138-142,140-140:19,19:2,2:0(30-102),0(30-102):0.5,0.5:.,.

Child:
chr19 47047398 . CAGCCTCGCCCTTCTTTTCCTTCAAATGCCGCCATCTCCTACCGAGTATGGCCTGGGCCAATCCCATCCATGTCCTACCGAGTATGGCCTGGGCCAATCCCACCCACGTCCGTCCCCATTCACGTCCTTTACAAACAGCCC . 0 . TRID=chr19_47047398_47047538_CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC;END=47047538;MOTIFS=CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC;STRUC=(CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC)n GT:AL:ALLR:SD:MC:MS:AP:AM 0/0:140,140:138-141,140-140:14,14:2,2:0(30-102),0(30-102):0.5,0.5:.,.

best

[tmokveld]

Hi thank you for your interest!

Regarding the empty output, trgt-denovo will produce an empty output if it encounters any problem whatsoever at a given locus.

That said, the warning you encountered, Skipping invalid data in M, is unusual. I ran a test by creating VCFs with your sample data and running trgt-denovo on some of my own BAM data, but could not reproduce the warning, where I got expected output:

trid	genotype	denovo_coverage	allele_coverage	allele_ratio	child_coverage	child_ratio	mean_diff_father	mean_diff_mother	father_dropout_prob	mother_dropout_prob	allele_origin	denovo_status	per_allele_reads_father	per_allele_reads_mother	per_allele_reads_child	father_dropoutmother_dropout	child_dropout	index	father_MC	mother_MC	child_MC	father_AL	mother_AL	child_AL	father_overlap_coverage	mother_overlap_coverage
chr19_47047398_47047538_CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC	0	0	8	0.0	16	0.0	0.0	0.0	0.0	0.0	?	X	8,8	8,8	8,8	N	N	N	02,2	2,2	2,2	140,140	140,140	140,140	7,7	7,7
chr19_47047398_47047538_CCATGTCCTACCGAGTATGGCCTGGGCCAATCCCAC	0	0	8	0.0	16	0.0	0.0	0.0	0.0	0.0	?	X	8,8	8,8	8,8	N	N	N	12,2	2,2	2,2	140,140	140,140	140,140	7,7	7,7

Could you share which TRGT version you have used? Additionally (if possible) would you be willing to share the VCFs and BAMs you are using? I can also be reached at [email protected]).

[BrendaLee1]

Hi,
Thank you for your reply. I tried trgt-v1.3.0-x86_64-unknown-linux-gnu and now I test trgt-v1.4.0-x86_64-unknown-linux-gnu and not finished yet. For the bam data, you mean spaning bam or whole genome bam data?

[tmokveld]

Great! Yes just the spanning BAM. Easiest would be to just include the reads spanning the problematic region (using samtools view, or run TRGT on only one region)

[BrendaLee1]

Hi,
I attached vcf and bam data below:
trgt_test.tar.gz
trgt version: 1.4.0

When I use trgt-denovo 0.2.0, I got empty output file and Skipping invalid data warning.

Best.

[tmokveld]

Thanks I just had a look at it and it seems that the TRGT BAM files have malformed reads. Specifically the rq fields seem to have an invalid type rq:d:-1 (d is not supported in the BAM spec). This is supposed to be rq:f:-1.0.

There should be an update of TRGT to 1.4.1 soon, I'll let you know when it is released.

[tmokveld]

TRGT v1.4.1 was just released (https://github.com/PacificBiosciences/trgt/releases/tag/v1.4.1).

Would you be able to try this version and see if it fixes the problem?

[BrendaLee1]

Thank you for your efforts, The new version fixed the problem.
I notice that the datasets you provided didn't include some of tandem repeat region we interested，I wonder wether the TRGT tool can generate repeat definition file for our own repeat region?

[tmokveld]

To create your own repeat definitions, an option you could try is tr-solve (https://github.com/trgt-paper/tr-solve).