A Label-Free, Sensitive, Real-Time, Semiquantitative Electrochemical Measurement Method for DNA Polymerase Amplification (ePCR)

Oligonucleotide hybridization to a complementary sequence that is covalently attached to an electrochemically active conducting polymer (ECP) coating the working electrode of an electrochemical cell causes an increase in reaction impedance for the ferro-ferricyanide redox couple. We demonstrate the use of this effect to measure, in real time, the progress of DNA polymerase chain reaction (PCR) amplification of a minor component of a DNA extract. The forward primer is attached to the ECP. The solution contains other PCR components and the redox couple. Each cycle of amplification gives an easily measurable impedance increase. Target concentration can be estimated by cycle count to reach a threshold impedance. As proof of principle, we demonstrate an electrochemical real-time quantitative PCR (e-PCR) measurement in the total DNA extracted from chicken blood of an 844 base pair region of the mitochondrial Cytochrome c oxidase gene, present at ∼1 ppm of total DNA. We show that the detection and semiquantitation of as few as 2 copies/μL of target can be achieved within less than 10 PCR cycles

XYalign: Inferring and Correcting for Sex Chromosome Ploidy in Next-Generation Sequencing Data

Sex chromosome aneuploidies are currently estimated to be as common as 1/400 in humans. Atypical ploidy will affect variant calling and measures of genomic variation that are central to most clinical genomic studies. Further, the high degree of similarity between gametologous sequences on the X and Y chromosomes can lead to the misalignment of sequencing reads and substantially affect variant calling. Here we present XYalign, a new tool that (1) quickly infers sex chromosome ploidy in NGS data (DNA and RNA), (2) remaps reads based on the inferred sex chromosome complement of the individual, and (3) outputs quality, depth, and allele-balance metrics across the sex chromosomes