DOI: 10.5061/dryad.3gm8c
title: Data from: A next-generation sequencing approach to river biomonitoring using benthic diatoms

description: Diatoms are main bioindicators used to assess the ecological quality of
 rivers, but their identification is difficult and time-consuming. Next
 Generation Sequencing (NGS) can be used to study communities of
 microorganisms, so we carried out a test of the reliability of 454
 pyrosequencing for estimating diatom inventories in environmental samples.
 We used small subunit ribosomal deoxyribonucleic acid (SSU rDNA),
 ribulose-1, 5-bisphosphate carboxylase (rbcL), and cytochrome oxidase I
 (COI) markers and examined reference libraries to define thresholds
 between the intra- and interspecific and intra- and intergeneric genetic
 distances. Based on tests of 1 mock community, we used a threshold of 99%
 identity for SSU rDNA and rbcL sequences to study freshwater diatoms at
 the species level. We applied 454 pyrosequencing to 4 contrasting
 environmental samples (with one in duplicate), assigned taxon names to
 environmental sequences, and compared the qualitative and quantitative
 molecular inventories to those obtained by microscopy. Species richness
 detected by microscopy was always higher than that detected by
 pyrosequencing. Some morphologically detected taxa may have been
 persistent frustules from dead cells. Some taxa detected by molecular
 analysis were not detected by morphology and vice versa. The main source
 of divergence appears to be inadequate taxonomic coverage in DNA reference
 libraries. Only a small percentage of species (but almost all genera) in
 morphological inventories were included in DNA reference libraries. DNA
 reference libraries contained a smaller percentage of species from
 tropical (27.1–38.1%) than from temperate samples (53.7–77.8%). Agreement
 between morphological and molecular inventories was better for species
 with relative abundance &gt;1% than for rare species. The rbcL marker
 appeared to provide more reproducible results (94.9% species similarity
 between the 2 duplicates) and was very useful for molecular
 identification, but procedural standardization is needed. The
 water-quality ranking assigned to a site via the Pollution Sensitivity
 diatom index was the same whether calculated with molecular or
 morphological data. Pyrosequencing is a promising approach for detecting
 all species, even rare ones, once reference libraries have been developed.
description: LACL1Benthic sample from Lay
 river on Mainland France that usually displays high diversity of diatom
 taxa. Sample collected by scraping material from the surface of stones
 taken from this river following the French standard (AFNOR 2007) used in
 routine biomonitoring programs. After DNA extraction and amplification of
 three markers (SSU rDNA, rbcL and CO1), we sequenced the fragmented PCR
 products in one random direction a GS FLX Titanium PicoTiterPlate 454
 (Roche, Basel, Switzerland) according to the manufacturer’s instructions.
 Reads were removed if they were too short, contained ambiguous bases, or
 showed low complexity when examined using PyroCleaner software version
 1.0.Pool1.RL3.clean.sff_extract.fastaBDNABenthic sample from Saint-Denis river on French tropical islands in the Indian Ocean (la Réunion) that usually displays low diversity of diatom taxa. Sample collected by scraping material from the surface of stones taken from this river following the French standard (AFNOR 2007) used in routine biomonitoring programs. After DNA extraction and amplification of three markers (SSU rDNA, rbcL and CO1), we sequenced the fragmented PCR products in one random direction a GS FLX Titanium PicoTiterPlate 454 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Reads were removed if they were too short, contained ambiguous bases, or showed low complexity when examined using PyroCleaner software version 1.0.Pool1.RL7.clean.sff_extract.fastaLACL2Benthic sample from Lay river on Mainland France that usually displays high diversity of diatom taxa. Sample collected by scraping material from the surface of stones taken from this river following the French standard (AFNOR 2007) used in routine biomonitoring programs. After DNA extraction and amplification of three markers (SSU rDNA, rbcL and CO1), we sequenced the fragmented PCR products in one random direction a GS FLX Titanium PicoTiterPlate 454 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Reads were removed if they were too short, contained ambiguous bases, or showed low complexity when examined using PyroCleaner software version 1.0.Pool1.RL4.clean.sff_extract.fastaCHLOBenthic sample from Chiers river on Mainland France that usually displays low diversity of diatom taxa. Sample collected by scraping material from the surface of stones taken from this river following the French standard (AFNOR 2007) used in routine biomonitoring programs. After DNA extraction and amplification of three markers (SSU rDNA, rbcL and CO1), we sequenced the fragmented PCR products in one random direction a GS FLX Titanium PicoTiterPlate 454 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Reads were removed if they were too short, contained ambiguous bases, or showed low complexity when examined using PyroCleaner software version 1.0.Pool1.RL5.clean.sff_extract.fastaCOINBenthic sample from Coconi river on French tropical islands in the Indian Ocean (Mayotte) that usually displays high diversity of diatom taxa. Sample collected by scraping material from the surface of stones taken from this river following the French standard (AFNOR 2007) used in routine biomonitoring programs. After DNA extraction and amplification of three markers (SSU rDNA, rbcL and CO1), we sequenced the fragmented PCR products in one random direction a GS FLX Titanium PicoTiterPlate 454 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Reads were removed if they were too short, contained ambiguous bases, or showed low complexity when examined using PyroCleaner software version 1.0.Pool1.RL6.clean.sff_extract.fasta