PASV: Automatic protein partitioning and validation using conserved residues

Abstract

Background: Increasingly, researchers use protein-coding genes from targeted PCR amplification or direct metagenomic sequencing in community and population ecology. Analysis of protein-coding genes presents different challenges from those encountered in traditional SSU rRNA studies. Most protein-coding sequences are annotated based on homology to other computationally-annotated sequences, which can lead to inaccurate annotations. Therefore, the results of sensitive homology searches must be validated to remove false-positives and assess functionality. Multiple lines of in silico evidence can be gathered by examining conserved domains and residues identified through biochemical investigations. However, manually validating sequences in this way can be time consuming and error prone, especially in large environmental studies. Results: An automated pipeline for protein active site validation (PASV) was developed to improve validation and partitioning accuracy for protein-coding sequences, combining multiple sequence alignment with expert domain knowledge. PASV was tested using commonly misannotated proteins: ribonucleotide reductase (RNR), alternative oxidase (AOX), and plastid terminal oxidase (PTOX). PASV partitioned 9,906 putative Class I alpha and Class II RNR sequences from bycatch in a global viral metagenomic investigation with >99% true positive and true negative rates. PASV predicted the class of 2,579 RNR sequences in >98% agreement with manual annotations. PASV correctly partitioned all 336 tested AOX and PTOX sequences. Conclusions: PASV provides an automated and accurate way to address post-homology search validation and partitioning of protein-coding marker genes. Source code is released under the MIT license and is found with documentation and usage examples on GitHub at https://github.com/mooreryan/pasv.

Ubiquitous, B12-dependent virioplankton utilizing ribonucleotide triphosphate reductase demonstrate interseasonal dynamics and associate with a diverse range of bacterial hosts in the pelagic ocean

“…Reads were translated into predicted amino acid sequences using a custom frameshift polishing pipeline (https://github.com/dnasko/frameshift_polisher). Key catalytic residues within translated amplicons were used for validating sequences as true RTPRs using the Protein Active Site Validation (PASV) (https://github.com/mooreryan/pasv) pipeline 55 . Detailed methods describing bioinformatic processing of RTPR amplicon sequences are provided in Supplementary Methods .…”

Novel Viral DNA Polymerases From Metagenomes Suggest Genomic Sources of Strand-Displacing Biochemical Phenotypes

“…To filter sequence hits, any query sequence hitting a UniProt DNA polymerase type-A family in the homology search was checked for conserved residues with Protein Active Site Validation (PASV, version 2.0.1, Moore et al, 2021 ), in multiple sequence alignment (MSA) mode using Clustal Omega version 1.2.4 (default settings), and E. coli DNA polymerase I, strain K12 (UniProt acc. P00582) as the reference sequence.…”