Sanger Sequencing for Variant Confirmation

Candidate variants were confirmed by Sanger sequencing using whole-genome amplified DNA from P2. Whole-genome amplification of genomic DNA was performed using the REPLI-g Midi Kit (Qiagen, Redwood City, CA, USA). PCR primers were designed using the Primer3 program v0.4.0 [29 (link), 30 (link)] and are listed in Supplementary Data 2. DNA fragments were amplified using HotStarTaq DNA Polymerase (Qiagen, Redwood City, CA, USA), purified using ExoSAP-IT PCR Purification Kit (USB Corporation, Cleveland, OH, USA), and sequenced using the Big Dye Terminator v3.1 kit (Applied Biosystems, Foster City, CA, USA). Sanger sequencing was performed on an ABI3130 Sequencer (Applied Biosystems), and visualised in Geneious 5.6.2 software (BioMatters Ltd., Auckland, New Zealand).

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Lung M.S., Mitchell C.A., Doyle M.A., Lynch A.C., Gorringe K.L., Bowtell D.D., Campbell I.G, & Trainer A.H. (2020). Germline whole exome sequencing of a family with appendiceal mucinous tumours presenting with pseudomyxoma peritonei. BMC Cancer, 20, 369.

Publication 2020

REPLI-g Midi Kit HotStarTaq DNA Polymerase ExoSAP-IT PCR Purification Kit Big Dye Terminator v3.1 kit ABI3130 Sequencer

Dna polymerase Genome Primers Redwood

Corresponding Organization :

Other organizations : Peter MacCallum Cancer Centre, Victorian Comprehensive Cancer Centre, University of Melbourne

Top 5 similar protocols

Variable analysis

independent variables

Whole-genome amplification of genomic DNA using the REPLI-g Midi Kit (Qiagen, Redwood City, CA, USA)

dependent variables

Candidate variants confirmed by Sanger sequencing

control variables

DNA fragments amplified using HotStarTaq DNA Polymerase (Qiagen, Redwood City, CA, USA)
DNA fragments purified using ExoSAP-IT PCR Purification Kit (USB Corporation, Cleveland, OH, USA)
Sanger sequencing performed on an ABI3130 Sequencer (Applied Biosystems)

controls

No positive or negative controls were explicitly mentioned.

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