Complete <i>F</i>. <i>heteroclitus</i> mitochondrial genome.

<p>Fragments (amplicons) used to PCR amplify the mitochondrial sequence. The primers used for <i>F</i>. <i>majalis</i> were designed using <i>F</i>. <i>heteroclitus</i> as a template.</p

Primers utilized to amplify the complete mitochondrial genome.

<p>Primers utilized to amplify the complete mitochondrial genome.</p

A Cost-Effective Approach to Sequence Hundreds of Complete Mitochondrial Genomes

<div><p>We present a cost-effective approach to sequence whole mitochondrial genomes for hundreds of individuals. Our approach uses small reaction volumes and unmodified (non-phosphorylated) barcoded adaptors to minimize reagent costs. We demonstrate our approach by sequencing 383 <i>Fundulus sp</i>. mitochondrial genomes (192 <i>F</i>. <i>heteroclitus</i> and 191 <i>F</i>. <i>majalis</i>). Prior to sequencing, we amplified the mitochondrial genomes using 4–5 custom-made, overlapping primer pairs, and sequencing was performed on an Illumina HiSeq 2500 platform. After removing low quality and short sequences, 2.9 million and 2.8 million reads were generated for <i>F</i>. <i>heteroclitus</i> and <i>F</i>. <i>majalis</i> respectively. Individual genomes were assembled for each species by mapping barcoded reads to a reference genome. For <i>F</i>. <i>majalis</i>, the reference genome was built <i>de novo</i>. On average, individual consensus sequences had high coverage: 61-fold for <i>F</i>. <i>heteroclitus</i> and 57-fold for <i>F</i>. <i>majalis</i>. The approach discussed in this paper is optimized for sequencing mitochondrial genomes on an Illumina platform. However, with the proper modifications, this approach could be easily applied to other small genomes and sequencing platforms.</p></div

Maximum likelihood phylogenetic reconstructions.

<p>A) Global phylogenetic tree produced using 5,088 sites dispersed throughout the entire mtDNA of most samples sequenced in this study. B) Gene tree for the CytB gene. Sequences CG1 through CG192 (<i>F</i>. <i>heteroclitus</i> samples) clustered with the <i>F</i>. <i>heteroclitus</i> CytB NCBI sequence (blue cartoon). Similarly, samples CG193 through CG384 (<i>F</i>. <i>majalis</i> samples) clustered with the <i>F</i>. <i>majalis</i> CytB NCBI sequence (red cartoon). C) Just as for CytB, sequenced samples form clades with their respective reference COI sequence from NCBI in the case of <i>F</i>. <i>heteroclitus</i> and from BOLD system v3 in the case of <i>F</i>. <i>majalis</i>.</p

Quality control information for <i>Fundulus majalis</i> samples.

<p>A) Average coverage depth along the mtDNA genome. Amplification products (amplicons) are shown across the top. B) Amplified view of the average, <i>F</i>. <i>majalis</i> mitochondrial genome, coverage depth. C) Percent of samples (out of 191) showing non-N nucleotides. D) Percent of samples showing different coverage depths. E) Percent of samples showing different mapping quality levels.</p

Quality control measurements of raw reads for <i>F</i>. <i>heteroclitus</i> (A, C, E) and <i>F</i>. <i>majalis</i> (B, D, F).

<p>A-B) Figure shows number of all available reads after trimming <i>vs</i>. read size. Nucleotide window analyses of <i>F</i>. <i>heteroclitus</i> aligned reads (C) and unaligned reads (E). Nucleotide window analyses of <i>F</i>. <i>majalis</i> aligned reads (D) and unaligned reads (F). The outer ribbon of low opacity represents the 10^th and 90^th quality percentiles. The inner opaque ribbon represents the upper and lower quality quartiles. Black lines represent averages.</p

Base composition across all sequences.

<p>Regular calls (A,C,T,G, or N) in A) <i>F</i>. <i>heteroclitus</i>, and C) <i>F</i>. <i>majalis</i>. Degenerate calls (W,S,M,K,Y) in B) <i>F</i>. <i>heteroclitus</i>, and D) <i>F</i>. <i>majalis</i>. </p

Library Preparation Costs.

<p>Library Preparation Costs.</p

Joint distribution of locus type and presence of clinal pattern for at least one allele based on broken-stick fits.

<p>Joint distribution of locus type and presence of clinal pattern for at least one allele based on broken-stick fits.</p

Identification of outlier loci based on Genomic Co-Co plot (left panel) and fdist2 (right panel) analyses.

<p>Contours of 75, 95 and 99% are shown on the co-co plot. Mean F<i>_ST</i> is dotted and 99% C.I. lines are in black on the fdist2 panel. Outlier loci are in red (95% C.I.), non-outlier loci are in grey, and Ldh allozyme and SNPs are blue.</p