Phylogenetic inference using ND2 sequence data.

<p>Maximum-likelihood tree inferred from ND2 sequence alignment of the formalin-fixed sample (MVZ 214979), the Anocar2.0 reference genome (Anocar2.0), four <i>Anolis carolinensis</i> collected from Louisiana, USA, eight other <i>Anolis</i> species, and <i>Oplurus cyclurus</i>. <i>A</i>. <i>carolinensis</i> image printed under a CC BY license with permission of the original photographer and copyright owner J. Losos.</p

Coverage of the mitochondrial genome.

<p>Distribution of read counts (in 10 bp bins) and depth of the mitochondrial genome from the MVZ 214979. A vertebrate mitochondrial map is used for reference on the bottom to label regions of protein coding and rRNA genes. The control region is at the end of the map and is not labelled.</p

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing

<div><p>For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.</p></div

Nuclear coverage.

<p>(a) Read count and depth in 10 Kbp bins along the length of the six largest <i>A</i>. <i>carolinensis</i> chromosomes (green bars) using the MVZ 214979 library. The green line indicates a read count of 100, and coverage of 1X. (b) Read count and depth is shown in 1 Kb bins along a randomly selected 5 Mbp segment of chromosome 1 using the MVZ 214979 library.</p

Patterns of mismatches in MVZ 214979 sequences.

<p>The frequencies of the 12 types of mismatches (y-axis) are plotted as a function of distance from the 5′ and 3′ ends of the sequence reads (x-axis). The frequencies of each mismatch type are coded in different colors and line patterns. ‘After cleaning’ shows mismatch frequencies after deleting the first 50 bp form the end of each read.</p

AbyssAssemblies

Contains the ABySS assembly output for each library. Each file has the following naming convention: LIBRARY_KMER_KMERCOV-contigs.f

Passerines_Alignments

Contains alignments for the Passerine samples

Passerines_CleanReads

Contains the cleaned reads for the Passerine samples

Enyalius_CleanReads

Contains cleaned reads for the Enyalius samples

References

Contains the references recovered for each locus for each sample