Next-generation sequencing (NGS) of DNA targeted amplicons was performed as previously described7 (link). In summary, the first PCR amplified the genomic site of interest with primers containing Illumina adapter sequences (Supplementary Table 9 ), and the second PCR added barcodes with primers containing unique pairs of p5/p7 Illumina barcodes that are analogous to TruSeq CD indexes. The libraries were pooled based on Pico Green (Promega) measurements on a BioTek Synergy HT plate reader, and the final pool was sequenced paired-end (PE) 2×150 on a MiSeq machine using 300-cycle MiSeq Reagent Kit v2 (Illumina). Demultiplexed FASTQs were downloaded from Basespace (illumina) and analyzed using the batch version of CRISPResso216 .
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Targeted NGS Amplicon Sequencing Protocol
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Other organizations : Massachusetts General Hospital, Center for Cancer Research
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Variable analysis
independent variables
- Primers containing Illumina adapter sequences used in the first PCR to amplify the genomic site of interest
- Demultiplexed FASTQ files generated from the sequenced paired-end (PE) 2×150 libraries
- Sequencing performed on a MiSeq machine using 300-cycle MiSeq Reagent Kit v2 (Illumina)
- Libraries pooled based on Pico Green (Promega) measurements on a BioTek Synergy HT plate reader
- Second PCR adding barcodes with primers containing unique pairs of p5/p7 Illumina barcodes that are analogous to TruSeq CD indexes
- Not explicitly mentioned
- Not explicitly mentioned
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