- Follow the standard Illumina procedures for Cluster Generation starting with 10 nM of the purified PCR product from Step 6. The final samples can contain up to 24 indexed libraries for sequencing on either the Illumina NextSeq or HiSeq instrument.
- Add 6 µl of each of the custom sequencing primers into the sequencing mix as directed by the Illumina NGS protocol:
- Read 1 Seq Primer
- Index 1 Seq Primer
- Read 2 Seq Primer
- Perform Illumina NGS Paired-End (PE) sequencing (75 cycles) using the Illumina NextSeq or HiSeq. Proceed to cluster amplification using the Illumina Paired-End Cluster Generation Kit; refer to the manufacturer’s instructions for this step. The optimal seeding concentration for cluster amplification of DNA libraries is approximately 6 to 8 pM.
Step Primer Cycles Read 1: Read 1 Seq Primer 60 Index 1: Index 1 Seq Primer 6 Index 2: Read 2 Seq Primer 6 Read 2: Read 2 Seq Primer 13
- NGS FASTQ sequencing files should be analyzed for quality using FastQC. Remove PCR duplicate reads, then map sequences to the appropriate reference genome. During development of this assay, we used reference repetitive element annotation files from the UCSC genome browser and annotated the GRCh38 (hg38) reference genome in CLC Genomics Workbench (CLC Bio/QIAGEN, Cambridge MA). Generate sequencing profiles of L1 insertions from cancer samples and matched normal controls as the reference to isolate all de novo cancer-specific L1 insertions.
Last modified: Mar 07, 2017
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