Bisulfite amplicon sequencing can detect Glia and Neuron cell-free DNA in blood plasma.

Sampling the live brain is difficult and dangerous, and withdrawing cerebrospinal fluid is uncomfortable and frightening to the subject, so new sources of real-time analysis are constantly sought. Cell-free DNA (cfDNA) derived from glia and neurons offers the potential for wide-ranging neurological disease diagnosis and monitoring. However, new laboratory and bioinformatic strategies are needed. DNA methylation patterns on individual cfDNA fragments can be used to ascribe their cell-of-origin. Here we describe bisulfite sequencing assays and bioinformatic processing methods to identify cfDNA derived from glia and neurons. In proof-of-concept experiments we describe the presence of both glia- and neuron-cfDNA in the blood plasma of human subjects following mild trauma. These detection of glia- and neuron-cfDNA represents a significant step forward in the translation of liquid biopsies for neurological diseases. Bisulfite amplicon sequencing libraries were created from neuron (n=13) and glia gDNA (n=7) from samples used for HM450 analysis (above) and PBMC (n=11) from Breachers training day 1, and cfDNA (n=47) from Breachers training days 1, 7, 8 and 9 following the protocols in Supplementary Material I. Libraries were denatured following Illumina protocols and sequenced on either the Illumina MiSeq using 2 x 26bp sequencing or the Illumina HiSeq2500 using 2 x 50bp sequencing.