phyloFlash: Rapid Small-Subunit rRNA Profiling and Targeted Assembly from Metagenomes

Abstract

The small-subunit rRNA (SSU rRNA) gene is the key marker in molecular ecology for all domains of life, but it is largely absent from metagenome-assembled genomes that often are the only resource available for environmental microbes. Here, we present phyloFlash, a pipeline to overcome this gap with rapid, SSU rRNA-centered taxonomic classification, targeted assembly, and graph-based binning of full metagenomic assemblies. We show that a cleanup of artifacts is pivotal even with a curated reference database. With such a filtered database, the general-purpose mapper BBmap extracts SSU rRNA reads five times faster than the rRNA-specialized tool SortMeRNA with similar sensitivity and higher selectivity on simulated metagenomes. Reference-based targeted assemblers yielded either highly fragmented assemblies or high levels of chimerism, so we employ the general-purpose genomic assembler SPAdes. Our optimized implementation is independent of reference database composition and has satisfactory levels of chimera formation. phyloFlash quickly processes Illumina (meta)genomic data, is straightforward to use, even as part of high-throughput quality control, and has user-friendly output reports. The software is available at https://github.com/HRGV/phyloFlash (GPL3 license) and is documented with an online manual. IMPORTANCE To track organisms across all domains of life, the SSU rRNA gene is the gold standard. Many environmental microbes are known only from high-throughput sequence data, but the SSU rRNA gene, the key to visualization by molecular probes and link to existing literature, is often missing from metagenome-assembled genomes (MAGs). The easy-to-use phyloFlash software suite tackles this gap with rapid, SSU rRNA-centered taxonomic classification, targeted assembly, and graph-based linking to MAGs. Starting from a cleaned reference database, phyloFlash profiles the taxonomic diversity and assembles the sorted SSU rRNA reads. The phyloFlash design is domain agnostic and covers eukaryotes, archaea, and bacteria alike. phyloFlash also provides utilities to visualize multisample comparisons and to integrate the recovered SSU rRNAs in a metagenomics workflow by linking them to MAGs using assembly graph parsing.

Torix Rickettsia are widespread in arthropods and reflect a neglected symbiosis

“…The SRA dataset [ 39 ] containing 1 individual from 1,341 arthropod species was screened with phyloFlash [ 40 ] using default parameters, which finds, extracts, and identifies SSU rRNA sequences. Reconstructed full 16S rRNA sequences affiliated to Rickettsia were extracted and compared to sequences derived from the targeted screen phylogenetically (see sections above) to assess group representation within the genus.…”

“…a single dataset per species was examined, and where multiple datasets existed for a species, that with the largest read count was retained. The resultant dataset [ 39 ], representing 1,341 arthropod species, was then screened with phyloFlash [ 40 ], which finds, extracts, and identifies single-subunit (SSU) ribosomal RNA (rRNA) sequences.…”

CaveThiovulaceaediffer metabolically and genomically from marine species

“…To assess the abundance of Thiovulum in the different environments of the cave, phyloFlash (Gruber-Vodicka et al , 2020) was used to identify and annotate 16S rRNA reads from the raw metagenomic data. Thiovulum was found in highest abundance (sequence frequency) in Air Bell 2 (35 %), followed by the Lake Room (5 %) and submerged microbial mats (0.9 %) (see pie charts in Fig.…”

“…To obtain information on relative Thiovulum abundance, the raw short-read libraries (amplicon and metagenomic) were analyzed with phyloFlash (V 3.3; (Gruber-Vodicka et al , 2020)).…”

Anaerobic endosymbiont generates energy for ciliate host by denitrification

“…For the ciliate phylogeny, sequences obtained from Sanger sequencing of picked ciliates were added to the EukRef-Ciliphora 30 Plagiopylea subgroup alignment using MAFFT 79 online service version 7 (argument:--addfragments). An additional metagenome-assembled full-length 18S rRNA gene sequence assigned to Plagiopylea was obtained using phyloFlash 80 v.3.0 from one of the Lake Zug metagenomes (MG_18_C) and also added to the alignment (argument:--add). A phylogenetic tree was calculated using IQ-TREE webserver ( http://iqtree.cibiv.univie.ac.at ) running IQ-TREE 81 1.6.11 with default settings and automatic substitution model selection (best-fit model: TIM2+F+I+G4).…”