Genomic DNA Sequences from Mastodon and Woolly Mammoth Reveal Deep Speciation of Forest and Savanna Elephants
(a) Mastodon shotgun 454 sequencing. We ligated 454-adaptors (green and blue) to the ends of the DNA molecules and sequenced the libraries on a Roche 454 GS. (b) Bioinformatic analysis of shotgun 454 sequences. To identify proboscidean sequence, we compared the sequences to databases consisting of the savanna elephant draft genome (loxAfr1), the human genome (hg18), the mouse genome (mm8), NCBI's nucleotide database of environmental samples (env), and NCBI's non-redundant nucleotide database (nr). The 454 sequences with a best match to loxAfr1 (in red) were aligned to loxAfr1. Alignments of at least 90 bp in length and with a similarity higher than 87% were used for primer design after filtering out known repeat elements (using the UCSC RepeatMasker database). Primers were based on loxAfr1 sequence flanking the mastodon sequence. (c) Multiplex PCR and sequencing of the targeted loci in modern elephants and mammoth. We show the protocol for the first of four rounds of the project (Table S3 provides details of the further rounds). A total of 213 primer pairs were randomly divided into 5 multiplex primer mixes with 41–44 primer pairs per mix. These mixes were used for the first step of the two-step multiplex PCR approach, for each of the 5 samples (La, Loxodonta africana; Lc, L. cyclotis; Em 1, Elephas maximus 1; Em 2, E. maximus 2; Mp, Mammuthus primigenius). Dilutions of these products were used as templates to amplify the loci individually in the second step (shown for L. africana), resulting in 213 distinct products per sample. These products were quantified, normalized, and merged into one pool per sample. A 454 library was prepared and sequenced on 1/16th of a picotiter plate of a Roche 454 GS.