Abstract
The recent rise in speed and efficiency of new sequencing technologies have facilitated high-throughput sequencing, assembly and analyses of genomes, advancing ongoing efforts to analyze genetic sequences across major vertebrate groups. Standardized procedures in acquiring high quality DNA and RNA and establishing cell lines from target species will facilitate these initiatives. We provide a legal and methodological guide according to four standards of acquiring and storing tissue for the Genome 10K Project and similar initiatives as follows: four-star (banked tissue/cell cultures, RNA from multiple types of tissue for transcriptomes, and sufficient flash-frozen tissue for 1 mg of DNA, all from a single individual); three-star (RNA as above and frozen tissue for 1 mg of DNA); two-star (frozen tissue for at least 700 μg of DNA); and one-star (ethanol-preserved tissue for 700 μg of DNA or less of mixed quality). At a minimum, all tissues collected for the Genome 10K and other genomic projects should consider each species’ natural history and follow institutional and legal requirements. Associated documentation should detail as much information as possible about provenance to ensure representative sampling and subsequent sequencing. Hopefully, the procedures outlined here will not only encourage success in the Genome 10K Project but also inspire the adaptation of standards by other genomic projects, including those involving other biota.
Highlights
The recent rise in speed and efficiency of new sequencing technologies have facilitated high-throughput sequencing, assembly and analyses of genomes, advancing ongoing efforts to analyze genetic sequences across major vertebrate groups
Review Advances in sequencing technology over the last decade [1,2,3] have made it feasible to acquire a database for genomes of 10,000 species of vertebrates, analogous to the Human Genome Project
Proper collection and preservation of tissues across vertebrate species is fundamental to establishing cell cultures and isolating nuclear and mitochondrial DNA, RNA, and potentially proteomes suitable for genomics, as these materials are susceptible to rapid post-mortem degradation or degradation following tissue-sampling from living specimens
Summary
The G10K project will attempt to follow the standards outlined (Table 1; Figure 1): **** (tissue stored in liquid nitrogen for at least 1 mg of DNA, isolated RNA and cell line/tissue cultures), *** (frozen tissue for at least 1 mg of DNA and isolated RNA), ** (frozen tissue for 700 μg DNA), and * (tissue preserved in ethanol for less than 700 μg DNA). * collections from rare species where sampling may be difficult will still be useful for initial wholegenome sequencing attempts. These guidelines can be extended to projects focusing on invertebrates (e.g., i5K [75]), plants, and fungi through similar permit, transport, and storage procedures, and considerations where species identification is difficult (e.g., barcoding and archiving procedures). Some ethical considerations may not be relevant (e.g., animal use protocols in invertebrates are restricted to cephalopods) and specialized protocols for tissue collection (e.g., animals with smaller body sizes) and establishment of viable cultures (e.g., plants) may differ. Additional file 2: A sample protocol for freezing tissue biopsy samples prior to subsequent initiation of cell culture. Competing interests The authors declare that they have no competing interests
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