Single molecule sequencing of the M13 virus genome without amplification

<div><p>Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.</p></div

Read length distribution after length and repeat filters (blue bars) and after alignment (red bars).

<p>Read length distribution after length and repeat filters (blue bars) and after alignment (red bars).</p

Sample preparation and sequencing process for single molecule sequencing of biological samples.

<p>Sample preparation and sequencing process for single molecule sequencing of biological samples.</p

Coverage depth distribution.

<p>(A) Coverage depth for each base on M13 reference. The average coverage depth is 316x±96x. (B) Coverage rate as a function of coverage depth.</p

M13 genome sequencing statistics.

<p>M13 genome sequencing statistics.</p