Petabase-scale sequence alignment catalyses viral discovery - Nature

Classification of assembled RdRP sequences. In brief, we defined a barcode sequence, the polymerase palmprint ,as an approximately 100-amino-acid segment of the RdRP palm subdomain delineated by well-conserved catalytic motifs. We implemented an algorithm, Palmscan, to identify palmprint sequences and discriminate RdRPs from reverse transcriptases.

Point errors are single-letter substitution and indel errors that may be caused by PCR or sequencing per se. Random point errors are not reproduced in multiple non-PCR duplicate reads and are unlikely to assemble because such errors almost always induce identifiable structures in the assembly graph that are pruned during graph simplification. In rare cases, a contig may contain a read with random point errors.

Chimeras of polymerases from different species could arise from PCR amplification or assembly. We used the UCHIME2 algorithmto screen assembled palmprint sequences, finding no high-scoring putative chimeras. Mosaic sequences formed by joining a polymerase to unrelated sequence would either have an intact palmprint, in which case the mosaic would be irrelevant to our analysis, or would be rejected by Palmscan owing to the lack of delimiting motifs.

Reverse transcriptases are homologous to RdRP. Retroviral insertions into host genomes induce ubiquitous sequence similarity between host genomes and viral RdRP. Palmscan was designed to discriminate RdRP from sequences of reverse transcriptase origin. Testing on a large decoy set of non-RdRP sequences with recognizable sequence similarity showed that the Palmscan false discovery rate for RdRP identification is 0.001.

Endogenous viral elements cannot be distinguished from viral genomes on the basis of the palmprint sequence alone. To assess the frequency of EVEs in our data, we re-assembled 890 randomly chosen libraries yielding one or more palmprints using all reads, extracted the 23,530 resulting contigs with a positive palmprint hit by Palmscan, and classified them using Virsorter2 .

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