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We annotated the predicted genes using the hmmsearch program from the HMMER package (version: 3.1b2)  in combination with the Pfam database v31 . We kept the matches exceeding the internal gathering threshold and presenting an independent e-value < 1e-5 and coverage > 0.4. In addition, we took in account multi-domain annotations and we removed overlapping annotations when the overlap is larger than 50%, keeping the ones with the smaller e-value. In addition, we took into account their order of occurrence on the sequence. We assigned a Pfam annotation to 44% of the initial ~322 million genes (Table 1).
|Original dataset||Annotated ORFs||Not-annotated ORFs|
Table 1. Annotated genes with Pfam.
The next step in the workflow is deep clustering of the genes.
The input for the functional annotation script hmmsearch_pfam.sh is the multi-fasta file containing the predicted ORFs sequences (amino acids). The output is a tab-separated file called domain hits table, for more information check the HMMER user guide. This table is then parsed to remove overlapping hits and to filter out the matches showing an iE-value > 1e-05 and a coverage < 40%. This is done calling the script hmmsearch_res_parser.sh and passing it the result table and the E-value and coverage parameters/values.
An example of the script usage can be found here:
"hmmsearch_pfam.sh": - input: "data/gene_prediction/TARA_OSD_GOS_malaspina_hmpI-II.fasta.gz" - output: "data/pfam_annotation/marine_hmp_pfam31_results.tsv" "hmmsearch_res_parser.sh": - input: "data/pfam_annotation/marine_hmp_pfam31_results.tsv", e-value=1e-05, coverage=0.4 - output: "data/pfam_annotation/marine_hmp_pfam31_1e-5_c04.tsv"
 Finn, R. D., Clements, J., & Eddy, S. R. (2011). HMMER web server: interactive sequence similarity searching. Nucleic Acids Research, 39(Web Server issue), W29–W37.
 El-Gebali, S., Mistry, J., Bateman, A., Eddy, S. R., Luciani, A., Potter, S. C., Qureshi, M., Richardson, L. J., Salazar, G. A., Smart, A., Sonnhammer, E. L. L., Hirsh, L., Paladin, L., Piovesan, D., Tosatto, S. C. E., & Finn, R. D. (2019). The Pfam protein families database in 2019. Nucleic Acids Research, 47(D1), D427–D432.