Open wangzhennan14 opened 4 years ago
Hi @wangzhennan14,
Sorry for the slow response, I was on holidays. Can you run grep the alignment record for that read (cce11e44-9f88-45f8-8fa4-5fdf23314323
) and paste it here?
Thanks, Jared
Hi @jts , The alignment record for the read(cce11e44-9f88-45f8-8fa4-5fdf23314323) was error.read.log Please check it and help me to solve this problem, thank you very much! wangzhennan
Hi @wangzhennan14,
That link appears to be broken.
Jared
Hi @jts , Sorry fo that broken link, I have reload the error reads as follow, error.read.log Please help me to solve this problem. There are many samples meeting this problem. Thank you very much! wangzhennan
Hi,
This read is very long (>400kb), and some aligners may emit invalid CIGAR strings for such long reads. Can you let me know:
Thanks, Jared
Hi @jts ,
Thanks. Can you send me the BAM record for this read? The SAM file looks ok but the CIGAR problem only happens in BAM.
Jared
Hello, was this ever solved? I've been running into the same problem lately. Does it seem to be related to a specific version of samtools or minimap2? Running samtools 1.9 and minimap 2.17-r974-dirty.
I don't think I ever received a bam file that allowed me to reproduce the problem, so haven't fixed it yet.
What's the easiest way I can send you mine? FYI, this is coming from FAF09701 of the human genome data.
Can you send me the read ID? I have that data here
8982dd2b-fddd-4c21-99b8-906d4bf06afb
This read works for me, when I download it using wget http://s3.amazonaws.com/nanopore-human-wgs/rel4-nanopore-wgs-4249180049-FAF09701.fastq.gz
. I'm using:
samtools 1.10
Using htslib 1.10.2
Copyright (C) 2019 Genome Research Ltd.
minimap2 2.17-r941
The basecalls in the fastq above are quite old, I haven't tried with newer basecalls.
Hmm...let me update samtools and let you know what happens.
Updated to samtools 1.12 and minimap2 2.18-r1015 Still the same error: Error: spliced alignments detected when loading read 8982dd2b-fddd-4c21-99b8-906d4bf06afb Please align the reads to the genome using a non-spliced aligner
Thoughts?
Could you provide the fastq and bam file containing this single read? You should be able to attach it here, or email to jared.simpson@oicr.on.ca.
Hi @jts,
I'm facing the same error as above using NGMLR v0.2.7 (-x ont --bam-fix) and nanopolish (v0.13.2 and latest build from master)
It happened the first time and is very rare, samples were sequenced using the SQK-ULK001 kit.
I extracted the read and made a package of fast5, fastq and bam to reproduce the error.
The steps to reproduce are (reference is mm10):
tar -xf spliced_read.tar.gz
nanopolish index -d ./ spliced_read.fastq
nanopolish call-methylation -r spliced_read.fastq -g mm10.fa -b spliced_read.bam
The output is:
chromosome strand start end read_name log_lik_ratio log_lik_methylated log_lik_unmethylated num_calling_strands num_motifs sequence Error: spliced alignments detected when loading read 7a521bd6-133e-4d07-9bae-0cf81dd031ee Please align the reads to the genome using a non-spliced aligner
The read cigar does not seem to have N's though.
samtools view spliced_read.bam | awk '($6 ~ /N/)'
Thank you for the great software and help,
Pay
Thanks, I'm on vacation now but will try with your data when I return to work. Could you try the methylation_bam branch, which uses a more recent htslib, to rule out an htslib issue?
Awesome, that fixed it!
I'm glad to hear that, thanks for reporting back so quickly. I'll try to merge in the htslib upgrade soon.
Hi, When I used nanopolish call methylation, there was an error as follow: Error: spliced alignments detected when loading read cce11e44-9f88-45f8-8fa4-5fdf23314323 Please align the reads to the genome using a non-spliced aligner comand.sh: line 3: 8436 Segmentation fault (core dumped) nanopolish call-methylation -t 50 -r all_NanoReads.fastq -b output.sorted.bam -g ref.fasta > methylation_calls.tsv But I used minimap2 to align reads vs reference as guided(https://nanopolish.readthedocs.io/en/latest/quickstart_call_methylation.html): minimap2 -a -t 35 -x map-ont ref.fasta all_NanoReads.fastq | samtools sort -T tmp -o output.sorted.bam What was the matter? Please give me some advice to solve this problem, thank you very much! Best wishes, Wang