Determining mitochondrial genome sequences from formalin fixed paddlefish (Polyodon spathula) samples

Abstract:

When studying molecular phylogenetics of fishes, we often encounter difficulty in collecting fresh samples of rare species; in the meaning time there are a large number of specimens stored in museum collections that could not be used due to formalin treatment on those specimens. For example, the Acipenseriformes. In order to study taxonomy and get the mitochondrial genome of formalin-fixed sturgeon specimens, we tested a new strategy of determining mitochondrial genome sequence from formalin-fixed samples. We treated muscle sample of paddlefish (Polyodon spathula) with formalin for different length of time, 1 hour, 1 day, 3 days, 10 days, 30 days and 150 days. We then extracted DNA from those samples using an ancient DNA method, captured and sequenced the mitochondrial genome of all samples using homemade biotinylated baits and Illumina sequencing. The ratio of on-target reads was above 45% for all samples. The genome sequences assembled are 16 524 bp in length, with 100% coverage and zero error rate. We also studied the relationship between DNA fragmentation and time used for formalin treatment. The results showed that when the length of the target amplicon was 650 bp, it could be amplified only in sample treated with formalin for 1-hour . When the length of the amplicons was 41 bp, 129 bp or 305 bp, a trace of product could be amplified for samples treated with formalin for up to 150 days. According to Q-ratio scores (the ratio between different size amplicon and the 41 bp amplicon), we found that the longer of formalin fixation time was, the lower Q-ratio score became. In other word, the longer of formalin-fixed time was, the greater degree of DNA fragmentation occurred. These results can augment usefulness of DNA extraction from formalin-fixed samples in molecular studies.