This normalization step corrected for differences in capture efficiency or recovery of Hsp90 protein and enabled data from multiple independent capture preparations to be compared.
All amplified libraries (pre-capture preparation with NEBNext or ThruPLEX) were purified with Agencourt AMPure XP beads (cat. no A63881) and quantified for sequencing using 2100 Bioanalyzer High sensitivity kit (cat. no 5067-4626).
The new method consists of two major steps: 1) large-insert capture library preparation, and 2) PacBio library preparation using the captured product as template.
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Pro-cues ought not to capture this preparation effect, as participants would have the duration of post-array interval (1900 ms), to engage in any process that could not be completed before the array, before having to make a response.
A detailed protocol for 6 Kb insert capture library preparation is included in Additional file 1.
We are also grateful to the advice and assistance given by Karen Roberston and Yvonne Bisset (Department of Dermatology, University of Edinburgh) regarding the photographic capture and preparation of the digital images.
Target capture, library preparation, and deep sequencing of the 30 normal/tumor paired frozen DNA samples were performed by the Oklahoma Medical Research Foundation Next Generation DNA Sequencing Core Facility (Oklahoma City, OK, USA).
For 1 Kb, 4 Kb and 8 Kb insert capture library preparation, parameters for adjusting the protocol are given in Table 1.
The data capture and preparation of the manuscript have followed PRISMA guidelines (refer to Additional Files 1 and 2 for the PRISMA checklist and flowchart).
