Generating high-quality plant and fish reference genomes from field-collected specimens by optimizing preservation

Abstract

Sample preservation often impedes efforts to generate high-quality reference genomes or pangenomes for Earth's more than 2 million plant and animal species due to nucleotide degradation. Here we compare the impacts of storage methods including solution type, temperature, and time on DNA quality and Oxford Nanopore long-read sequencing quality in 9 fish and 4 plant species. We show 95% ethanol largely protects against degradation for fish blood (22 °C, ≤6 weeks) and plant tissue (4 °C, ≤3 weeks). From this furthest storage timepoint, we assemble high-quality reference genomes of 3 fish and 2 plant species with contiguity (contig N50) and completeness (BUSCO) that achieve the Vertebrate Genome Project benchmarking standards. For epigenetic applications, we also report methylation frequency compared to liquid nitrogen control. The results presented here remove the necessity for cryogenic storage in many long read applications and provide a framework for future studies focused on sampling in remote locations, which may represent a large portion of the future sequencing of novel organisms.

Fig. 1. Experimental approach: comparing impacts of storage conditions on DNA QC, sequencing, and assembly metrics.

a Nine fish species and four plant species were collected and b preserved using various storage conditions for up to 6 weeks (fish) or 3 weeks (plants). c HMW DNA extraction methods with slight modifications. d Workflow for assessing impacts of storage conditions on DNA QC and shallow sequencing performance. A subset of species (indicated by color coding) at their storage extremes were fully sequenced and assembled. Figure was made using Biorender.

Fig. 2. DNA fragment length predicted read length and assembly contiguity from deep sequencing.

a Femto Pulse DNA fragment length distributions, b deep sequencing read N50 length, and c assembly contig N50 length presented in the same sequence as Table 1: M. californiensis (orange), G. nigericans (purple), K. azureus (blue), M. esculenta (green), S. bicolor (turquoise).