Sticky problems: extraction of nucleic acids from molluscs

Abstract

Traditional molecular methods and omics-techniques across molluscan taxonomy increasingly inform biology of Mollusca. Recovery of DNA and RNA for such studies is challenged by common biological properties of the highly diverse molluscs. Molluscan biomineralization, adhesive structures and mucus involve polyphenolic proteins and mucopolysaccharides that hinder DNA extraction or copurify to inhibit enzyme-catalysed molecular procedures. DNA extraction methods that employ the detergent hexadecyltrimethylammoniumbromide (CTAB) to remove these contaminants importantly facilitate molecular-level study of molluscs. Molluscan pigments may stain DNA samples and interfere with spectrophotometry, necessitating gel electrophoresis or fluorometry for accurate quantification. RNA can reliably be extracted but the ‘hidden break’ in 28S rRNA of molluscs (like most protostomes) causes 18S and 28S rRNA fragments to co-migrate electrophoretically. This challenges the standard quality control based on the ratio of 18S and 28S rRNA, developed for deuterostome animals. High-AT content in molluscan rRNA prevents the effective purification of polyadenylated mRNA. Awareness of these matters aids the continuous expansion of molecular malacology, enabling work also with museum specimens and next-generation sequencing, with the latter imposing unprecedented demands on DNA quality. Alternative methods to extract nucleic acids from molluscs are available from literature and, importantly, from communications with others who study the molecular biology of molluscs. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

Chromosome‐level reference genome for European flat oyster (Ostrea edulis L.)

“…Bivalve genome assembly has classically been hampered by genetic complexities including high heterozygosity and repeat content (Davison & Neiman, 2021 ), along with the challenge of extracting pure high molecular weight DNA (Adema, 2021 ). However, recent advances in long‐read sequencing technologies have enabled high‐quality genome sequences to be generated for multiple bivalves, including C. gigas (Peñaloza et al, 2021 ; Qi et al, 2021 ), the scallop Pecten maximus (Kenny et al, 2020 ) and hard clam Mercenaria mercenaria (Farhat et al, 2022 ; Song et al, 2021 ).…”

Chromosome level reference genome for European flat oyster (Ostrea edulis L.)

“…Bivalve genome assembly has classically been hampered by genetic complexities including high heterozygosity and repeat content (Davison & Neiman, 2021), along with the challenge of extracting pure high-molecular weight DNA (Adema, 2021). However, recent advances in long-read sequencing technologies have enabled high quality genome sequences to be generated for multiple bivalves, including C. gigas (Peñaloza et al, 2021; Qi et al, 2021), the scallop Pecten maximus (Kenny et al, 2020) and hard clam Mercenaria mercenaria (Song et al, 2021; Farhat et al, 2022).…”

Pearls of wisdom—a Theo Murphy issue on molluscan genomics

“…The directions outlined in these papers may directly shape decisions regarding project design (e.g. how to best extract DNA [2] and which genomic technologies to use [3]). They should also inspire research aimed at testing hypotheses posed and questions raised by our authors.…”

“…Now, the quality of the output mainly depends upon the input material (DNA), the bioinformatic methods that are used to extract the data and assemble the genomes, and the platforms upon which we store and make the data accessible for (re)use. This Theo Murphy issue therefore opens with a timely review on the extraction of nucleic acids from molluscs [2], the value of which was noted by a referee as ‘very rare, yet of extremely high value: the collection of informal knowledge, which is essentially fed by experiments and experiences that, because they were unsuccessful, never made it into official literature’.…”