Hi All, I am looking for a way to visualize my RNA reads from Nanopore Direct RNA sequencing on IGV. I sequenced an IVT reaction, and use minimap2 to map the fastq files with my own specific sequence (fasta file). I got the bam files. Next step I would look for the variant transcripts and visualize them on IGV with my own reference sequences. How could I do that? Thank you.
Thank you, I did sort and index bam file, but still it did not work :( I loaded on IGV and get this error:
Error loading BAM file: htsjdk.samtools.SAMException: Sequence name 'saRNA_ref\' doesn't match regex: '[0-9A-Za-z!#$%&+./:;?@^_|~-][0-9A-Za-z!#$%&+./:;=?@^_|~-]'
How should I fix it? Thanks a lot.
Hi, I used EPI2ME Desktop Agent with the option of Fastq Custom Alignment to generate the bam file. I uploaded my reference sequence (fasta file) to that tool. And that exact fasta file was used to upload to IGV to create the custom genome. I am not sure what I did wrong :( If you know, please let me know. Thank you!!
Should be fairly easy if you've got this far, sorting and indexing the bam is easy with samtools via bioconda.
Then https://software.broadinstitute.org/software/igv/LoadGenome
Remember to look for transcript annotation which fits your fasta sequence.
I would also use genome wide annotation eg gencode etc and map to the genome too.
Thank you, I did sort and index bam file, but still it did not work :( I loaded on IGV and get this error:
Error loading BAM file: htsjdk.samtools.SAMException: Sequence name 'saRNA_ref\' doesn't match regex: '[0-9A-Za-z!#$%&+./:;?@^_|~-][0-9A-Za-z!#$%&+./:;=?@^_|~-]'
How should I fix it? Thanks a lot.
Sounds like the BAM header is broken. Check the header using
samtools view -h x.bam | less
Compare to other examples, and or paste the output of this here, it might contain special characters
samtools faidx x.fasta
samtools view -h x.bam | less
Result:
@HD VN:1.6 SO:coordinate
@SQ SN:saRNA_ref\ LN:9383
@RG ID:none
@PG PN:minimap2 ID:minimap2 VN:2.17-r941 CL:minimap2 -y -a -x map-ont -t 1 --MD /tmp/datasets/1bcf05961acd0e52b280d1e1a4e3cd8e5cbdc19b/reference_multi_index_8G.fa ./FAK73268_pass_cf5085a8_0-0002.fastq
@PG ID:samtools PN:samtools PP:minimap2 VN:1.10 (pysam) CL:samtools sort --output-fmt BAM -@ 1 -o output/FAK73268_pass_cf5085a8_0-0002.fastq.bam output/FAK73268_pass_cf5085a8_0-0002.fastq.sam
samtools faidx x.fasta
[fai_build_core] different line length in sequence '(null)'.
Could not build fai index /Users/TrinhTat/Documents/Trinh_HM2022/Research/RNA_Core/Nanopore/072122_IVT/saRNA_ref.fa.fai
It generated saRNA_ref.fa.fai but may have error, since the message said it could not build fai index. Could you please show me to build this index with a fasta file just containing my sequence of interest. Thank you very much!!
How did you make /Users/TrinhTat/Documents/Trinh_HM2022/Research/RNA_Core/Nanopore/072122_IVT/saRNA_ref.fa? Is there more than one sequence in this file?
Can you show us the output of grep "^>" /Users/TrinhTat/Documents/Trinh_HM2022/Research/RNA_Core/Nanopore/072122_IVT/saRNA_ref.fa?
grep "^" saRNA_ref.fa > output head output
{\rtf1\ansi\ansicpg1252\cocoartf2638
\cocoatextscaling0\cocoaplatform0{\fonttbl\f0\fswiss\fcharset0 Helvetica;}
{\colortbl;\red255\green255\blue255;}
{\*\expandedcolortbl;;}
\margl1440\margr1440\vieww51000\viewh27180\viewkind0
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f0\fs24 \cf0 >saRNA_ref\
TAATACGACTCACTATAATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACT
Here it is. Is it supposed to be correct? I made the fasta file using TextEdit with > on the first line, the second line is my sequence. Then I saved it as fasta file. Please let me know if I am doing things correctly. Thank you!!
Use a programmers editor (like NotePad++ on PC) or (BBEdit on macOS) and remove all of this stuff at the beginning of the file.
{\rtf1\ansi\ansicpg1252\cocoartf2638
\cocoatextscaling0\cocoaplatform0{\fonttbl\f0\fswiss\fcharset0 Helvetica;}
{\colortbl;\red255\green255\blue255;}
{\*\expandedcolortbl;;}
\margl1440\margr1440\vieww51000\viewh27180\viewkind0
\pard\tx720\tx1440\tx2160\tx2880\tx3600\tx4320\tx5040\tx5760\tx6480\tx7200\tx7920\tx8640\pardirnatural\partightenfactor0
\f0\fs24 \cf0
Also remove the trailing \ after reference name here: >saRNA_ref\
You may still need to end up redoing the entire process again with this edited file, if IGV does not like the edited file.
Hi, I did remove all the stuff as you suggested, head output
>saRNA_ref
TAATACGACTCACTATAATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTC
Loaded on IGV with the new edited fasta file as reference genome, and the sorted bam file, but I still got the error as below
/Users/TrinhTat/Documents/Trinh_HM2022/Research/RNA_Core/Nanopore/072122_IVT_aligned/PASS/bam_files/FAK73268_pass_combined.sorted.bam: An error occurred while accessing: /Users/TrinhTat/Documents/Trinh_HM2022/Research/RNA_Core/Nanopore/072122_IVT_aligned/PASS/bam_files/FAK73268_pass_combined.sorted.bam Error loading BAM file: htsjdk.samtools.SAMException: Sequence name 'saRNA_ref\' doesn't match regex: '[0-9A-Za-z!#$%&+./:;?@^_|~-][0-9A-Za-z!#$%&*+./:;=?@^_|~-]*'
Is the problem came from fasta file or bam file? Thank you!!
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