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. 2020 Oct 27;5(5):e00920-20.
doi: 10.1128/mSystems.00920-20.

phyloFlash: Rapid Small-Subunit rRNA Profiling and Targeted Assembly from Metagenomes (V体育平台登录)

Harald R Gruber-Vodicka #  1 , Brandon K B Seah #  2 , Elmar Pruesse  3
Affiliations

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

Harald R Gruber-Vodicka et al. mSystems. .

"VSports" 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 VSports手机版. 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. .

Keywords: SSU; gene assembly; metagenomics; taxonomic profiling. V体育安卓版.

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"VSports在线直播" Figures

FIG 1
FIG 1
Flowchart of the phyloFlash pipeline. Gray boxes depict main data files, and white boxes with bold print show processes. Solid black arrows indicate primary workflow; dashed lines indicate processing of secondary output.
FIG 2
FIG 2
Database filtering removes low-complexity read hits. Informational redundancy (k = 5) in reads extracted by SortMeRNA (left) or BBmap (right) using unfiltered (above) and filtered (below) reference databases, for library with NCBI:SRA accession no. ERR315856.
FIG 3
FIG 3
False-positive read extraction depends on stringency cutoffs, but only BBmap results improve with read length. Origin of reads extracted by SortMeRNA and BBmap at different parameters (horizontal axes) from simulated metagenome of 100-bp paired-end (PE) reads (above) or 150-bp PE reads (below), showing the proportion of total originating from actual SSU rRNA features (left) and the origin of non-SSU rRNA-extracted reads (right). Horizontal lines indicate the total actual SSU rRNA origin reads in each library (20,213 and 13,829, respectively).
FIG 4
FIG 4
Mapper and cutoff settings influence SSU rRNA read extraction from Tara Oceans metagenome libraries but yield highly similar assembly ratios. (a) Fraction of reads extracted by both tools at different BBmap minimum identities (id.) (horizontal) and SortMeRNA E-value cutoffs (vertical). (b) Reads assembled by SPAdes versus total reads extracted for different parameter settings (colors) of SortMeRNA (top) and BBmap (bottom), overlaid with linear regression lines and 1:1 line for reference (gray).
FIG 5
FIG 5
Comparison of scaffolds assembled by SPAdes, Emirge, and Matam using reads extracted by SortMeRNA (E-value of 10−5, filtered reference database) from 85 Tara Oceans metagenomic libraries. (a) Plot of scaffolds assembled per library versus number of extracted SSU rRNA reads. (b) Histograms of per-scaffold metrics for each assembly tool: scaffold length (bp), undetermined bases (Ns) per scaffold, uchime_ref chimerism score, and identity to closest reference sequence.
FIG 6
FIG 6
Heatmap of taxonomic assignments (rows) for SSU rRNA reads in metagenome libraries of six gutless oligochaete species with symbiotic consortia (columns). The plot was generated with the comparison scripts provided with phyloFlash. Color intensities represent the percentage of reads mapping to a given taxon, separated by prokaryotes (blue) and eukaryotes (red).
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