Sample Preservation, DNA or RNA Extraction and Data Analysis for High-Throughput Phytoplankton Community Sequencing

Abstract

Phytoplankton is the basis for aquatic food webs and mirrors the water quality. Conventionally, phytoplankton analysis has been done using time consuming and partly subjective microscopic observations, but next generation sequencing (NGS) technologies provide promising potential for rapid automated examination of environmental samples. Because many phytoplankton species have tough cell walls, methods for cell lysis and DNA or RNA isolation need to be efficient to allow unbiased nucleic acid retrieval. Here, we analyzed how two phytoplankton preservation methods, three commercial DNA extraction kits and their improvements, three RNA extraction methods, and two data analysis procedures affected the results of the NGS analysis. A mock community was pooled from phytoplankton species with variation in nucleus size and cell wall hardness. Although the study showed potential for studying Lugol-preserved sample collections, it demonstrated critical challenges in the DNA-based phytoplankton analysis in overall. The 18S rRNA gene sequencing output was highly affected by the variation in the rRNA gene copy numbers per cell, while sample preservation and nucleic acid extraction methods formed another source of variation. At the top, sequence-specific variation in the data quality introduced unexpected bioinformatics bias when the sliding-window method was used for the quality trimming of the Ion Torrent data. While DNA-based analyses did not correlate with biomasses or cell numbers of the mock community, rRNA-based analyses were less affected by different RNA extraction procedures and had better match with the biomasses, dry weight and carbon contents, and are therefore recommended for quantitative phytoplankton analyses.

Directional High-Throughput Sequencing of RNAs Without Gene-Specific Primers

“…Our previous phytoplankton mock community study showed that RNA-based community profiling correlated better with the biomass measures than DNA-based sequencing, in which the variation in the rRNA gene copy numbers per algae cell can be over 100-fold [1]. Moreover, profiling microbial communities using 16S/18S rRNA instead of rRNA genes especially brings out living organisms with active protein synthesis and is free of dissolved or relic DNA [2].…”

“…and Chlorella pyrenoidosa . The biomass, dry mass, carbon content and cell number values of each species of the pool was determined earlier [1], but symbiotic prokaryotic cells were unidentified. Here, total RNA was extracted from the mock cells (a detailed protocol and list of the reagents is available in the Supplementary Protocol) using a Direct-Zol RNA MicroPrep isolation kit (Zymo Research, CA, USA).…”

“…Sequencing was performed with the Ion Torrent Personal Genome Machine (PGM) as described by Mki etal . [1]. The data was analyzed using CLC Genomics Workbench 11 software (www.qiagenbioinformatics.com) using a phytoplankton reference database comprising mock community sequences [1] or Silva v128 16S reference rRNA gene database for prokaryotic analysis and using OTU 97 identity clustering level (Supplementary Figure 2).…”

“…[1]. The data was analyzed using CLC Genomics Workbench 11 software (www.qiagenbioinformatics.com) using a phytoplankton reference database comprising mock community sequences [1] or Silva v128 16S reference rRNA gene database for prokaryotic analysis and using OTU 97 identity clustering level (Supplementary Figure 2). Relative abundances of strains were used to calculate Bray-Curtis similarity matrix, based on which nonmetric multidimensional scaling (NMDS) was calculated with 1000 repeats in PRIMER v. 6.1.12 and PERMANOVA+ v.1.0.2 (PRIMER-E/Quest Research Limited, Albany, New Zealand).…”

“… (A) Primer-free and gene-specific [1] sequencing of rRNA yielded different proportional relationships between species, where the results of the former better resembled the dry biomass and carbon content of the pool. Wet biomass [1] was determined microscopically.…”