Application of high-throughput sequencing (HTS) metabarcoding to diatom biomonitoring: Do DNA extraction methods matter?

Abstract

Current freshwater biomonitoring with diatoms is based on microscopic examination of the morphology of their silica skeleton. This standardized approach is time consuming and requires a high degree of taxonomic expertise. Metabarcoding combined with high-throughput sequencing (HTS) has great potential for next-generation biomonitoring applications but requires standardization. Molecular inventories are strongly influenced by the DNA extraction method used, but the effect of extraction protocols has not been tested to enable selection of the best DNA extraction method for HTS metabarcoding. We used 5 DNA extraction methods combining various types of cell lysis and DNA purification to extract DNA from 8 pure diatom cultures and 8 samples from streams and lakes with differing water quality. We compared the methods based on: 1) quality and purity of the extracted DNA, 2) community inventories obtained from HTS targeting the ribulose-1, 5-bisphosphate carboxylase (rbcL) barcode, and 3) similarity between molecular and microscopy-based inventories of community composition and the Specific Pollutionsensitivity Index [SPI]. A method based on GenElute ™ -LPA had higher extraction efficiency than the 4 commercial kits but had the highest polymerase chain reaction inhibition level. All 5 methods were efficient for HTS, and method did not affect operational taxonomic unit richness. We observed variations in the relative abundance of some taxa within Nitzschia, Amphora, Encyonema, Gomphonema, and Navicula between 2 of the 5 methods, but method did not affect global diatom community composition or SPI values. SPI values calculated from microscopy-based inventories and molecular inventories based on all 5 extraction methods were strongly correlated. For convenience purposes (high DNA quantity and low cost), we encourage standardization of HTS diatom biomonitoring based on the SA-Gen method.

Avoiding quantification bias in metabarcoding: Application of a cell biovolume correction factor in diatom molecular biomonitoring

“…From each cultivation replicate, 10 ml of culture was centrifuged at 17,000 × g for 30 min (Figure ). Total DNA was extracted from the resulting pellet using a protocol based on GenEluteTM‐LPA DNA precipitation (Sigma‐Aldrich, St Louis, Missouri) as previously described (Vasselon, Domaizon, et al., ). Then, qPCR assays were performed for each of the eight diatom species on DNA extracted at all seven sampling times and with each of the three replicates, using the QuantiTect SYBR Green PCR Kit (Life Technologies, Carlsbad, USA) and the Rotor‐Gene Q (Qiagen, Hilden, Germany).…”

“…Rimet, ). Different studies have already revealed the potential application of diatom metabarcoding in freshwater quality assessment (Apothéloz‐Perret‐Gentil et al., ; Kermarrec et al., ; Vasselon, Domaizon, Rimet, Kahlert, & Bouchez, ; Vasselon, Rimet, Tapolczai, & Bouchez, ; Visco et al., ). However, discrepancies between DNA metabarcoding and microscopy have been observed in species composition and relative abundance (Zimmermann, Glöckner, Jahn, Enke, & Gemeinholzer, ).…”

“…With respect to qualitative aspects, the incompleteness of the reference databases, the choice of the DNA marker and the efficiency of the PCR primers have been identified as important biases affecting species detection using DNA metabarcoding (Pawlowski, Lejzerowicz, Apotheloz‐Perret‐Gentil, Visco, & Esling, ). For benthic diatoms, the rbc L gene has proved to be an appropriate taxonomic marker for biomonitoring (Kermarrec et al., , ; Vasselon, Domaizon, et al., ; Vasselon, Rimet, et al., ) and a well‐curated barcode reference library is already available in open‐access to assign species names to rbc L sequences (R‐Syst::diatom, Rimet et al., ). However, no clear relationship has yet been demonstrated between the relative species abundances obtained by DNA metabarcoding with the rbc L barcode and those obtained by morphological observations (Rimet et al., ).…”

The impact of OTU sequence similarity threshold on diatom‐based bioassessment: A case study of the rivers of Mayotte (France, Indian Ocean)

“…High-throughput sequencing (HTS) technology, in combination with the aforementioned meta-barcoding, allows for simultaneously identifying multiple taxa from multiple environmental samples (Taberlet, Coissac, Pompanon, Brochmann, & Willerslev, 2012). The incorporation of molecular techniques in biomonitoring has caused remarkable progress over the past decade in terms of optimal genetic marker selection (Kermarrec et al, 2013), HTS platform (Loman et al, 2012;Shokralla, Spall, Gibson, & Hajibabaei, 2012), DNA extraction (Vasselon, Domaizon, Rimet, Kahlert, & Bouchez, 2017), and the bioinformatics required to analyze the HTS data. This facilitates expanding the sampling network to include more sites monitored on a more frequent basis and has thereby revolutionized the field of biomonitoring (Baird & Hajibabaei, 2012;Keck, Vasselon, Tapolczai, Rimet, & Bouchez, 2017).…”

The potential of High-Throughput Sequencing (HTS) of natural samples as a source of primary taxonomic information for reference libraries of diatom barcodes

“…25 mg of wet pellet was used for each sample. DNA extraction was based on the Sigma-Aldrich GenEluteTM-LPA DNA protocol which was used in previous studies Chonova et al 2016;Vasselon et al 2017). The final elution volume was 40 µl.…”