Since quite a few substantial scale sequencing projects are now directed in direction of exome sequencing tactics, the ques tion stays no matter if targeted re sequencing on FFPE tissue may well be attainable. We for that reason divided prostate tissue samples of radical prostatectomy specimens and stored a single portion as snap frozen tissue blocks, the other as FFPE material. We employed materials from both preservation technologies Panobinostat solubility for DNA extraction and subsequent hybridization for DNA capturing followed by Illumina sequencing from the complete Exome target area at the same time as to get a three. 9 Mb custom designed target area. 1 caveat of up coming generation sequencing protocols from FFPE materials may be the large temperature wanted to melt the paraffin, which ends in a significant fraction of single stranded DNA.
Even so, for the subsequent library preparation stage double stranded DNA is needed. We found that an first heating phase to 75 C for five min is sufficient to melt the paraffin and preserve dsDNA. For the two DNA components snap frozen at the same time as FFPE stored we identified an normal of 75% NVP-TAE226 price on the sequencing reads found inside the entire exome target area and even more than 99% of the regions have been captured by not less than one particular read. Each preservation technologies have equivalent cover age profiles and we found a high degree of correlation of enrichment per exon amongst experiments, that’s depicted as coefficient of variation. Even though the coefficient of variation is reduced within precisely the same planning technol ogy, e. g. snap frozen versus snap frozen and FFPE versus FFPE, the variation is larger amongst snap frozen and FFPE.
Consequently, 1 need to ideally continue to be inside the identical tissue planning engineering for a single set of experiments. It truly is regarded that FFPE tissues are susceptible to spontaneous deamination of guanine and cytosine during the tissue preservation process andor through storage and the Illumina sequencing technological innovation is biased for underrepresentation and lowered good quality at loci with excessive base composi tions. In this regard, we observed a slight, but not sig nificant, shift from the GC dependent coverage profile among the snap frozen and FFPE tissue. To assess the impact of coverage depth over the sensitivity and specificity of sequence variant detection, we used genotype calls of an Affymetrix SNP array 6. 0 through the cryo material and in contrast every place to your total exome sequencing information. For each tissue preparations we attained pretty comparable accuracies over 98%, even at cov erages down to 10. Subsequent we investigated the reproducibility of single nucleotide variation detection in snap frozen ver sus FFPE tissues. We identified 179 discordant loci investigating positions with no less than twenty fold coverage. The potential artifacts may be grouped into false posi tives, e.