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From our set of analyses, we have confirmed that both fragmentation and sequencing artefacts are common forms of DNA damage from formalin-fixed material even if blocks are relatively new.
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There was a significant difference for both fragmentation and sequence artefacts between anatomical pathology (AP) laboratories (p < 0.0001, Kruskal-Wallis test).
cDNA fragmentation and sequencing adaptor tagging war performed using Illumina Nextera XT kit (FC-131-1096).
The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories.
The two most intense ions in each MS1 scan were chosen for CID fragmentation and sequencing.
To our knowledge, this study represents the largest assessment of formalin induced fragmentation and sequencing artefacts using clinical FFPE samples.
In this study we explored two common form of DNA damage caused by formalin, i.e. fragmentation and sequencing artefacts.
Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing.
Fragmentation and sequencing of the peptides using MS/MS is usually an additional experimental step and is not the focus of this work.
This study assessed the prevalence of DNA fragmentation and sequencing artefacts from a large cohort of FFPE tumours using a uniform approach whereby all blocks were of similar age, were extracted in the same manner and were run through the same MPS platform.
As expected, the generation of multiply charged peptide ions greatly facilitated their fragmentation and thus sequencing.
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