No edges formed by TNF

[Sophie Adelberg]

Could someone help me figure out what is going wrong? It claims that the number of large contigs >= 2500 are 0 but this is not the case which can clearly be seen from my depth files (attached is the depth file for the sample I am trying to run below)

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\$ ~/anaconda3/bin/metabat2 -i ./idba/idba_contigs.fa/d11_1901Seekatz24_idba_contig.fa -a ./binning/d11_1901Seekatz24_depth.txt -o ./binning/d11_1901Seekatz24/bin -t 24 -v

MetaBAT 2 (2.15 (Bioconda)) using minContig 2500, minCV 1.0, minCVSum 1.0, maxP 95%, minS 60, maxEdges 200 and minClsSize 200000. with random seed=1599071389

[00:00:00] Executing with 24 threads

[00:00:00] Parsing abundance file

[00:00:00] Parsing assembly file

[00:00:00] Number of large contigs >= 2500 are 0. 

[00:00:00] Reading abundance file

[00:00:00] Finished reading 1068 contigs and 1 coverages from ./binning/d11_1901Seekatz24_depth.txt

[00:00:00] Number of target contigs: 0 of large (>= 2500) and 0 of small ones (>=1000 & <2500). 

[00:00:00] Start TNF calculation. nobs = 0

[00:00:00] Finished TNF calculation.                                  

[00:00:00] Finished Preparing TNF Graph Building [pTNF = 69.40]                                             

[00:00:00] Finished Building TNF Graph (0 edges) [15.2Gb / 251.6Gb]                                          

No edges were formed by TNF.

[00:00:00] Rescuing singleton large contigs

[00:00:00] There are 0 bins already

[00:00:00] Outputting bins

[00:00:00] -nan% (0 bases) of large (>=2500) and 0.00% (0 bases) of small (<2500) contigs were binned.

0 bins (0 bases in total) formed.

[00:00:00] Finished

[Rob Egan]

The contig length field in the abundance file are not used in determining the actual lengths of the sequences. I suspect the contigs file is malformed and for some reason metabat did not throw an error. Can you attach 1 or two sequences from this file too?

/idba/idba_contigs.fa/d11_1901Seekatz24_idba_contig.fa

[Sophie Adelberg]

Here are a few of the shortest ones

contig-125_196 length_204 read_count_83035

GGCGAATTGGAAATGACATACCCTGATGGCTCACAAGATGTAATGAAAGTTCCAGTGAAAGTAGGTACGGATGCCCAAGCCTTAACACCAAAAGTTAAGTCAAACTTAGAAGTTTCCTTAGGCTCTAAGTTGACCCCAGAAACGACATTAGATCCAAGTGTAGCTTTACCGACAGGAACTAAGCTGAATTGGAAGACACCAGTG

contig-125_197 length_203 read_count_244348

CACTGGGCCCCGTCAAGTAGACAATTAAATAATTAAAATCCTAGGCAGCCTGATTTCGGTATTCTTCCGGAGTTAGGCTGTTTAGTTTTGTTTGATAGCGTTGTGTATTATAGAACTTGATGTAAGCGTTGATAGCTTCGACTAGCTCTTCATATGTTTTATATTTCTTCCAGCTGTAACATTCAACCTTGAAGTGACTCCAA

contig-125_198 length_203 read_count_150760

AAGAAGTTTCGTTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCATCCTGAGCCAGGATCAAACTCTCAATTTAAAGTTTGTGACTCTTTATTTTTTACTAGCGAATTGACTTCGCAAAATGTTTATTGCTCTTATCACTTAGATAAGAGACCCTGCACATTTGCTTATCGAAACTTTGTTCAGTTTTCAAAGATCTACT

[Rob Egan]

These contig names to not match the depth file you provided. Please show me the first two sequences from ./idba/idba_contigs.fa/d11_1901Seekatz24_idba_contig.fa that should match the first two lines of the depth file, so that I can verify the files are not corrupted and that metabat can recognize the data. The log you showed indicates that metabat did not recognize the fasta file as containing any sequences.

c_000000000006  5663    27.9819 27.9819 34.109
c_000000000007  2878    3.80609 3.80609 3.67231

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[Sophie Adelberg]

The actual sequences are quite long but here are the header for the first 2 from this file:

contig-125_0 length_242649 read_count_173992025
contig-125_1 length_143136 read_count_83655600

Sorry, I am a grad student trying to come up with a pipeline for our lab to do metagenomic sequence analysis so I do not have a lot of experience

[Sophie Adelberg]

I found the issue, thanks for the help!

[Rob Egan]

Glad you found it. Was it a formatting issue in that fasta file? That is what I was suspecting and why I wanted to view the sequences that you were feeding it. If it was a formatting issue, I’ll see what I can do to have metabat output an informative error in the future.

[Sophie Adelberg]

Yes, it was. Thank you!