A Comparison and Integration of MiSeq and MinION Platforms for Sequencing Single Source and Mixed Mitochondrial Genomes

<div><p>Single source and multiple donor (mixed) samples of human mitochondrial DNA were analyzed and compared using the MinION and the MiSeq platforms. A generalized variant detection strategy was employed to provide a cursory framework for evaluating the reliability and accuracy of mitochondrial sequences produced by the MinION. The feasibility of long-read phasing was investigated to establish its efficacy in quantitatively distinguishing and deconvolving individuals in a mixture. Finally, a proof-of-concept was demonstrated by integrating both platforms in a hybrid assembly that leverages solely mixture data to accurately reconstruct full mitochondrial genomes.</p></div

SNP Phasing and Deconvolution in Two Loci.

<p>The selected loci contain proximate SNPs that are private to 005 and 047. A) Four SNPs in a 1kb window with ticks of 50 bp (np 3,800–4,800). B) Three SNPs in 300bp window with 50 bp ticks (np 11,200–11,500). Read counts for each of the SNPs are shown In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.t001" target="_blank">Table 1</a>. MiSeq and MinION mixture alignments are the top two tracks. Phasing and deconvoluted read sets make up the middle and bottom sets of tracks. The middle two tracks are the reads that represent 005 and the bottom two tracks are the reads that represent 047.</p

Mixture Concordance by VAF.

<p>The MiSeq single source 0.90 VAF call sets were combined for both 005 and 047 to inform the optimal MiSeq mixture comparison. The y-axis has MiSeq VAF SNP call sets for the single source (top) and mixture (bottom). On the top, the MiSeq mixture SNP call sets are plotted by VAF from 0.17 to 0.53 using increments of 0.02 across the x-axis and compared to the single source combined 0.90 VAF SNP set. The F1-Scores are equal between 0.23 and 0.29 in the mixture. On the bottom, the empirically determined VAF range (0.23 to 0.29) for the mixture MiSeq VAF permitted the MiSeq Mixture VAF to be set at 0.25 to compare with MinION VAFs ranging 0.17 to 0.53 using increments of 0.02. The highest F1-Score between the MiSeq (0.25) and MinION (0.45) VAFs are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.g004" target="_blank">Fig 4</a>.</p

Single Source Sample Concordance by VAF.

<p>Each single source sample (004, 005, and 047) is characterized by a heatmap, which compares SNP call sets between the two platforms. SNP call sets are plotted by VAF for both the MinION and MiSeq data from 0.05 to 0.95 using increments of 0.05. The MinION VAF is on the x-axis and the MiSeq VAF is across the y-axis. Concordance is determined by calculating the F1-Score using the MiSeq calls as the ground truth. The value of each F1-Score comparison is shown as increasingly darker shades of blue for higher values. The highest F1-Scores are shown for each of the source samples 004, 005, and 047 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.g003" target="_blank">Fig 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.s003" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.s004" target="_blank">S3</a> Figs, respectively.</p

Coverage and Concordance Circos for 004.

<p>The coverage depth per base is shown for the MiSeq (orange) and MinION (blue) shown on the outer ring using a log10 scale. The inner ring shows concordance at each SNP using a MiSeq VAF of 0.90 and a MinION VAF of 0.65. Text color denotes the categorization of each SNP under the VAF combination providing the highest concordance (F1-Score). Green text indicates true positives and black text is for false negatives present in the MinION call sets. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.s003" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167600#pone.0167600.s004" target="_blank">S3</a> Figs for similar plots of 005 and 047, respectively. Black arrows indicate the locations and orientations of the primers used for amplification.</p

Mitochondrial Depth of Coverage.

<p>Coverage distributions of MiSeq (orange) and MinION (blue) sequencing data are grouped together by sample across the x-axis. Depth observations are plotted against the y-axis with a log10 scale. The average distributions stem from the single source samples (004, 005, and 047) only.</p