Metabat2 shows error

Hello,

I am trying to perform binning with metabat2 but it is showing error ' the order of contigs in abundance file is not the same as the assembly file: k141_5762'.

I first sorted my BAM file (base) pbaranw@enggpz1p23:~/parul/output$ samtools sort mapped.bam -o sorted.bam

I checked if BAM files is sorted (base) pbaranw@enggpz1p23:~/parul/output$ samtools view -H sorted.bam

@HD VN:1.4 SO:coordinate

*I generated the depth file* (base) pbaranw@enggpz1p23:~/parul/output$ jgi_summarize_bam_contig_depths --outputDepth depth.txt sorted.bam Output depth matrix to depth.txt jgi_summarize_bam_contig_depths v2.15-6-g8b5702b 2021-10-17T15:10:38 Output matrix to depth.txt 0: Opening bam: sorted.bam Processing bam files WARNING: your aligner reports an incorrect NM field. You should run samtools calmd! nm < ins + del: cmatch=0 nm=2 ( insert=0 + del=60 + mismatch=2 == 62) A00405:433:HHYFCDSX2:3:1176:5321:1376 1:N:0:ACGATCAG+ACGGAACA Thread 0 finished: sorted.bam with 15775901 reads and 14529494 readsWellMapped Creating depth matrix file: depth.txt Closing most bam files Closing last bam file Finished

The codes for metabat2 I ran is: (base) pbaranw@enggpz1p23:~/parul/output$ metabat2 -i final.contigs.fa -a depth.txt -o bins

MetaBAT 2 (v2.15-6-g8b5702b) using minContig 2500, minCV 1.0, minCVSum 1.0, maxP 95%, minS 60, maxEdges 200 and minClsSize 200000. with random seed=1643665071 [Error!] the order of contigs in abundance file is not the same as the assembly file: k141_5762

Please any help. Thank you so much

metabat

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written 4.5 years ago by salmon •

1 answer

The problem is with this command:

jgi_summarize_bam_contig_depths --outputDepth depth.txt sorted.bam

In order to sort the contigs properly, it needs to know the assembly FASTA file from which the .BAM file was created. Like so:

jgi_summarize_bam_contig_depths --outputDepth depth.txt --referenceFasta assembly.fa sorted.bam

The rest should work once you fix the depth file.

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written 4.5 years ago by Mensur Dlakic

Thank you for quick reply.

I tried running this command but still error is showing

jgi_summarize_bam_contig_depths --outputDepth depth.txt --referenceFasta final.contigs.fa sorted.bam

I get following result:

Output depth matrix to depth.txt Reference fasta file final.contigs.fa jgi_summarize_bam_contig_depths v2.15-6-g8b5702b 2021-10-17T15:10:38 Output matrix to depth.txt Reading reference fasta file: final.contigs.fa ... 6008 sequences 0: Opening bam: sorted.bam Error: referenceFile: final.contigs.fa contig 0 is not the same as in the bam headers (bam reports k141_502 flag=1 multi=2.0000 len=306 with 306 len, reference loaded k141_4762 with 313 len)!

I created the BAM file with following code. should I use referenceFasta as g1_trimmed.fastq

bbmap.sh in= g1_trimmed.fastq out=mapped.bam

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written 4.5 years ago by salmon •

You need to map the reads to the same assembly file that will later be used by jgi_summarize_bam_contig_depths:

bbmap.sh ref=final.contigs.fa in=g1_trimmed.fastq out=mapped.bam nodisk

Next steps are to sort the files ( samtools sort mapped.bam -o sorted.bam ) and repeat the commands for depth file creation and metabat2.

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written 4.5 years ago by Mensur Dlakic

Thank you so much. It worked for me. However, three bins are created. As I am working with one sample (i.e forward and reverse read of one pure bacterial culture), I should get only one bin (Please correct me if I am wrong). Does creation of 3 bins shows the sign of contamination.

Any help appreciated.

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written 4.5 years ago by salmon •

Don't know why you were even binning the contigs coming from a single-species assembly, but apparently that is something worth doing.

Yes, you should get a single bin, with some caveats:

that your culture is truly pure
that your bacterium doesn't have an intracellular parasite
that you have removed contigs smaller than at least 1.5 kb, and it may be worth going up to 2 kb
that this bacterium doesn't have plasmids or other extrachromosomal DNA elements
that this bacterial genome does not have any funky repeats, pathogenic islands, or other sequence regions of different sequence properties from the rest of genome
that you have done binning properly

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written 4.5 years ago by Mensur Dlakic

Thank you so much for your explanation.

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written 4.5 years ago by salmon •