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bedtools getFasta doesn't match chromosomes error

I know this error has been reported numerous times and have read through proposed solutions such as presented in this Error In Bedtools Getfasta: Chromosome Not Found and none of them have resolved this error. I am still unable to get my file to successfully grab sequences.

I actually haven't had this issue before, used the exact same script the only change is using a newer genome file GRCm39 instead of GRCm38, but both were downloaded from ensembl using their FTP links. Whats interesting is that when I run the command with the GRCm38 it works but not with the GRCm39.

I have no idea why it wouldn't work for both? Is it the index file? I use bedtools to generate the .fai file.

Here is a comparison header of the files and they look similar:

Mus_musculus.GRCm38.dna.primary_assembly.fa
1 dna:chromosome chromosome:GRCm38:1:1:195471971:1 REF
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

Mus_musculus.GRCm39.dna.primary_assembly.fa
>1 dna:chromosome chromosome:GRCm39:1:1:195154279:1 REF
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

For sake of completion here are the rest of the important pieces I am using

Here are a couple lines of the error:

index file /blue/berglund/j.ellis/annotation_files/FTP_GenesAndSeqFiles/Mus_musculus.GRCm39.dna.primary_assembly.fa.fai not found, generating...
WARNING. chromosome (19) was not found in the FASTA file. Skipping.
WARNING. chromosome (12) was not found in the FASTA file. Skipping.

Here is my script:

bedtools getfasta -fo ./fasta_SEevents_Struct_MEF.fa -fullHeader -s\
 -fi /blue/berglund/j.ellis/annotation_files/FTP_GenesAndSeqFiles/Mus_musculus.GRCm39.dna.primary_assembly.fa\
 -bed ./BED_SEevents_Struct_MEF.txt

Here is a sample of my bed file:

19      55924529        55924580        Tcf7l2  1       +
19      53242503        53242627        Add3    1       +
19      45364086        45364274        Btrc    1       +

Of note, to generate this bed file I use a python script to convert a pandas df to a bed file and upload it to the cluster using cyberduck. Below is the export command

bed_out.to_csv('./BED_SEevents_Struct_MEF.bed', sep = '\t', index = False, header = False)
fasta getfasta bedtools
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updated by Ram Ram • written by Joseph • Joseph

Hi! Have you checked if your fasta file actually contains an entry for chromosome 19?

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written by iraun iraun

I just double checked using cat Mus_musculus.GRCm39.dna.primary_assembly.fa | sed -n /19/p > a.txt and in my out file I see it come up as expected.

>1 dna:chromosome chromosome:GRCm39:1:1:195154279:1 REF
>11 dna:chromosome chromosome:GRCm39:11:1:121973369:1 REF
>19 dna:chromosome chromosome:GRCm39:19:1:61420004:1 REF
>7 dna:chromosome chromosome:GRCm39:7:1:144995196:1 REF
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written by Joseph • Joseph

Thanks to Pierre Lindenbaum's comment for the help! I figure out a solution. Since the .fai files were not indexing the chromosomes properly for whatever reason using bedtools in-built .fai generation I utilized samtools faidx intead and was able to get an .fai file that pulled just the chromosome #s not the whole line. Then it ran fine!

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written by Joseph • Joseph

1 answer

what are the outputs of the following commands

file Mus_musculus.GRCm39.dna.primary_assembly.fa
file BED_SEevents_Struct_MEF.bed
cut -f 1 BED_SEevents_Struct_MEF.bed | uniq | sort | uniq | paste -s -d ';'
grep "^>" Mus_musculus.GRCm39.dna.primary_assembly.fa  | cut -c2- | cut -d ' ' -f1 | paste -s -d ';'
cut -f1  Mus_musculus.GRCm39.dna.primary_assembly.fa.fai   | paste -s -d ';'
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written by Pierre Lindenbaum Pierre Lindenbaum

In order:

Mus_musculus.GRCm39.dna.primary_assembly.fa: ASCII text

BED_SEevents_Struct_MEF.bed: ASCII text

1;10;11;12;13;14;15;16;17;18;19;2;3;4;5;6;7;8;9;X

(im shortening the output for the numbered chromosomes for brevity) 1;10;11;12;13;14;15;16;17;18;19;2;3;4;5;6;7;8;9;MT;X;Y

1 dna:chromosome chromosome:GRCm39:1:1:195154279:1 REF;10 dna:chromosome chromosome:GRCm39:10:1:130530862:1 REF;11 dna:chromosome chromosome:GRCm39:11:1:121973369:1 REF;12 dna:chromosome chromosome:GRCm39:12:1:120092757:1 REF;13 dna:chromosome chromosome:GRCm39:13:1:120883175:1 REF;14 dna:chromosome chromosome:GRCm39:14:1:125139656:1 REF;15 dna:chromosome chromosome:GRCm39:15:1:104073951:1 REF;16 dna:chromosome chromosome:GRCm39:16:1:98008968:1 REF;17 dna:chromosome chromosome:GRCm39:17:1:95294699:1 REF;18 dna:chromosome chromosome:GRCm39:18:1:90720763:1 REF;19 dna:chromosome chromosome:GRCm39:19:1:61420004:1 REF;2 dna:chromosome chromosome:GRCm39:2:1:181755017:1 REF;3 dna:chromosome chromosome:GRCm39:3:1:159745316:1 REF;4 dna:chromosome chromosome:GRCm39:4:1:156860686:1 REF;5 dna:chromosome chromosome:GRCm39:5:1:151758149:1 REF;6 dna:chromosome chromosome:GRCm39:6:1:149588044:1 REF;7 dna:chromosome chromosome:GRCm39:7:1:144995196:1 REF;8 dna:chromosome chromosome:GRCm39:8:1:130127694:1 REF;9 dna:chromosome chromosome:GRCm39:9:1:124359700:1 REF;MT dna:chromosome chromosome:GRCm39:MT:1:16299:1 REF;X dna:chromosome chromosome:GRCm39:X:1:169476592:1 REF;Y dna:chromosome chromosome:GRCm39:Y:1:91455967:1

here is the output using GRCm38: 1;10;11;12;13;14;15;16;17;18;19;2;3;4;5;6;7;8;9;MT;X;Y

So clearly the GRCm39 has a different naming scheme or there is something weird that happened when I tried to download that file. I used wget to grab the file. Not sure how to go from there?

Thank you for the reply!

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written by Joseph • Joseph

I figured it out with that help, my comment details that. Thank you very much!

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written by Joseph • Joseph

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