Profile hidden Markov model sequence analysis can help remove putative pseudogenes from DNA barcoding and metabarcoding datasets
AbstractBackgroundPseudogenes are non-functional copies of protein coding genes that typically follow a different molecular evolutionary path as compared to functional genes. The inclusion of pseudogene sequences in DNA barcoding and metabarcoding analysis can lead to misleading results. None of the most widely used bioinformatic pipelines used to process marker gene (metabarcode) high throughput sequencing data specifically accounts for the presence of pseudogenes in protein-coding marker genes. The purpose of this study is to develop a method to screen for obvious pseudogenes in large COI metabarcode datasets. We do this by: 1) describing gene and pseudogene characteristics from a simulated DNA barcode dataset, 2) show the impact of two different pseudogene removal methods on mock metabarcode datasets with simulated pseudogenes, and 3) incorporate a pseudogene filtering step in a bioinformatic pipeline that can be used to process Illumina paired-end COI metabarcode sequences. Open reading frame length and sequence bit scores from hidden Markov model (HMM) profile were used to detect pseudogenes.ResultsOur simulations showed that it was more difficult to identify pseudogenes from shorter amplicon sequences such as those typically used in metabarcoding (∼300 bp) compared with full length DNA barcodes that are used in construction of barcode libraries (∼ 650 bp). It was also more difficult to identify pseudogenes in datasets where there is a high percentage of pseudogene sequences. We show that existing bioinformatic pipelines used to process metabarcode sequences already remove some apparent pseudogenes, especially in the rare sequence removal step, but the addition of a pseudogene filtering step can remove more.ConclusionsThe combination of open reading frame length and hidden Markov model profile analysis can be used to effectively screen out obvious pseudogenes from large datasets. There is more to learn from COI pseudogenes such as their frequency in DNA barcode and metabarcoding studies, their taxonomic distribution, and evolution. Thus, we encourage the submission of verified COI pseudogenes to public databases to facilitate future studies.
