Targeted NGS was performed on patient genomic DNA using the Trusight Cancer sequencing panel (Illumina) that contains 1,736 genomic regions from 94 genes suspected of having a role in cancer predisposition, including the RB1 gene. An analytical validation of our panel has shown a sensitivity of 98.2%, specificity of 100%, and reproducibility of 99.5%. The gene coverage analysis on this panel revealed that exonic and flanking intronic regions of the RB1 gene (NM_000321 ) showed coverage of >99% (≥ 20 reads) with a mean read depth of 405X. The Nextera DNA library preparation protocol (Illumina) to convert input genomic DNA (gDNA) into adaptor-tagged indexed libraries was essentially performed as previously described [15 (link)]. The tagged and amplified sample libraries were checked for quality and they were quantified using the BioAnalyzer (Agilent). Up to 6–10 pM of the pooled library was loaded and sequenced on the MiSeq platform (Illumina), according to the manufacturer’s instructions.
Nextera dna library preparation protocol
Manufactured by Illumina
The Nextera DNA library preparation protocol is a laboratory equipment product designed for efficient and streamlined preparation of DNA libraries for next-generation sequencing. It provides a standardized workflow for DNA fragmentation, adapter ligation, and library amplification, enabling consistent and reproducible library construction.
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2 protocols using nextera dna library preparation protocol
1
Targeted NGS for Cancer Predisposition
2
Nextera DNA Library Preparation
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We used the Nextera DNA library preparation protocol (Illumina) to convert input genomic DNA (gDNA) into adapter-tagged indexed libraries, which was essentially performed as previously described. 17 The quality of tagged and amplified sample libraries was checked by using the BioAnalyzer (Agilent, Santa Clara, CA, USA). The pooled library was loaded and sequenced on the MiSeq platform (Illumina), according to the manufacturer's instructions.
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