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Cell free DNA (cfDNA) are pieces of DNA that are not enclosed in cells but are freely moving around in the blood stream. Their average length per piece of cfDNA is about 170 base pairs. The length of the reference human DNA is about 3.3 billion base pairs. The length and the location of these pieces of cfDNA in comparison to the reference human DNA can reveal the tissue of origin of cfDNA. Imagine that the reference human genome was a picture and cfDNA were pieces of a jigsaw puzzle of various sizes. The location at which you place the puzzle pieces would be important to get an accurate picture. In this project we wanted to identify if we could distinguish between cfDNA from individuals with FSHD when compared to unaffected individuals. CfDNA could be utilized as a biomarker in FSHD if the size and/or the location of these pieces are unique to FSHD.
There is low amount and high variability of cfDNA between samples. However, we successfully extracted cfDNA from blood samples of 12 individuals with FSHD, 4 unaffected individuals and from muscle cells grown in dishes. Using muscle cells in culture as a control allows testing for the changes in cfDNA when the muscle cells are provoked to produce DUX4, the protein toxic to muscles in FSHD. After extracting cfDNA we further processed the samples in a way that the contents could be readable utilizing a next generation sequencing machine. We then performed light sequencing on the samples for quality assurance in preparation for high cost deep sequencing. Light sequencing allowed us to obtain a snippet of the jigsaw puzzle that makes up the cfDNA from each individual sample. We are utilizing various software to place these readable pieces of cfDNA against the human reference DNA. Our future goal is to utilize Illumina’s HiSeq sequencing platform to perform deep sequencing. Deep sequencing would allow us to obtain many more readable pieces of cfDNA to get a much more accurate picture. In conclusion, we show here for the first time the extraction of cfDNA from individuals with FSHD. We have successfully prepared samples for sequencing and are set up to analyze the sequencing data.