No Reads assigned

Hi,

I have aligned reads to the nt database using bowtie2, and now I’m trying to do taxonomic binning using MEGAN6. However, I’m getting no assigned reads.

(base) [danielle.perley@buddy ancientDNA_test]$ ~/megan/tools/blast2rma -f SAM -i 18-144-001_qsorted.sam -mdb megan-nucl-Oct2019.db -o 18-144-001.rma6
Version MEGAN Community Edition (version 6.18.5, built 14 Feb 2020)
Copyright © 2019 Daniel H. Huson. This program comes with ABSOLUTELY NO WARRANTY.
Loading ncbi.map: 2,175,506
Loading ncbi.tre: 2,175,510
Processing SAM file: 18-144-001_qsorted.sam
Output file: 18-144-001.rma6
Classifications: Taxonomy
Generating RMA6 file Parsing matches
Annotating RMA6 file using FAST mode (accession database and first accession per line)
Parsing file 18-144-001_qsorted.sam
Parsing file: 18-144-001_qsorted.sam
10% 20% 30% 40% 50% 60% 70% 80% 90% 100% (91.6s)
Total reads: 182,403
Alignments: 4,929,529
100% (0.0s)
Binning reads: Initializing…
Initializing binning…
Using ‘Naive LCA’ algorithm for binning: Taxonomy
Binning reads…
Binning reads: Analyzing alignments
Total reads: 182,403
With hits: 19,630
Alignments: 4,929,529
Assig. Taxonomy: 0
MinSupport set to: 91
Binning reads: Applying min-support & disabled filter to Taxonomy…
Min-supp. changes: 0
Binning reads: Writing classification tables
Numb. Tax. classes: 2
Binning reads: Syncing
Class. Taxonomy: 2
100% (46.9s)
Total time: 145s
Peak memory: 1.9 of 2.1G

I get the same results using sam2rma.

First couple lines of my sam file:
(base) [danielle.perley@buddy ancientDNA_test]$ samtools view 18-144-001_qsorted.sam | head -n 2

M02569:185:000000000-C4LW3:1:1101:1891:13303 16 CP017316.1 6087214 255 75M * 0 0 GCGGCCGGCGTCACCGTCGGCGCCCTGCTGGCGCAGGACATGCTCAGCTCCACCAGCCTCGCGGGCCTGCCCAGC >+84++74,:,B++6++86+8+,++6++,C66C,;,@,E@6,7,C,<C@+++7B7@;:@;9DE?CCCC AS:i:-28 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:2C26C15G16C2A5G3 YT:Z:UU

M02569:185:000000000-C4LW3:1:1101:1891:13303 16 CP023696.1 6276501 2 75M * 0 0 GCGGCCGGCGTCACCGTCGGCGCCCTGCTGGCGCAGGACATGCTCAGCTCCACCAGCCTCGCGGGCCTGCCCAGC >+84++74,:,B++6++86+8+,++6++,C66C,;,@,E@6,7,C,<C@+++7B7@;:@;9DE?CCCC AS:i:-28 XS:i:-32 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:2C26C15G16C2A5G3 YT:Z:UU

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