A streamlined solution for processing, elucidating and quality control of cyclobutane pyrimidine dimer sequencing data

Abstract

UV radiation may lead to melanoma and nonmelanoma skin cancers by causing helix-distorting DNA damage such as cyclobutane pyrimidine dimers (CPDs). These DNA lesions, if located in important genes and not repaired promptly, are mutagenic and may eventually result in carcinogenesis. Examining CPD formation and repair processes across the genome can shed light on the mutagenesis mechanisms associated with UV damage in relevant cancers. We recently developed CPD-Seq, a high-throughput and single-nucleotide resolution sequencing technique that can specifically capture UV-induced CPD lesions across the genome. This novel technique has been increasingly used in studies of UV damage and can be adapted to sequence other clinically relevant DNA lesions. Although the library preparation protocol has been established, a systematic protocol to analyze CPD-Seq data has not been described yet. To streamline the various general or specific analysis steps, we developed a protocol named CPDSeqer to assist researchers with CPD-Seq data processing. CPDSeqer can accommodate both a single-and multiple-sample experimental design, and it allows both genome-wide analyses and regional scrutiny (such as of suspected UV damage hotspots). The runtime of CPDSeqer scales with raw data size and takes roughly 4 h per sample with the possibility of acceleration by parallel computing. Various guiding graphics are generated to help diagnose the performance of the experiment and inform regional enrichment of CPD formation. UV damage comparison analyses are set forth in three analysis scenarios, and the resulting HTML pages report damage directional trends and statistical significance. CPDSeqer can be accessed at https://github.com/shengqh/cpdseqer.

High UV damage and low repair, but not cytosine deamination, stimulate mutation hotspots at ETS binding sites in melanoma

“…After Illumina sequencing, CPD locations were identified by retrieving adjacent dinucleotides on the opposite strand upstream of the 5′ end of each Read1 ( Fig. 2 A ), similar to the original CPD-seq protocol ( 11 , 25 ).…”

ASH1L-MRG15 methyltransferase deposits H3K4me3 and FACT for damage verification in nucleotide excision repair

“…Following published protocols 5 , 6 a single library was obtained for each experimental condition. Sequencing reads were processed using custom scripts based on the CPDSeqer pipeline 74 . Raw reads were trimmed and adapters removed with bbduk (bbmap v.38.90) followed by alignment (bwa mem v.0.7.17-r1188) to the human reference genome build GCA hg38 lacking chrY but including non-canonical contigs.…”

“…Instead in ASH1L –/– cells, there were 46 million CPDs at 0 h and 45 million by 3 h. Raw dipyrimidine counts were binned into genomic regions of interest and transformed to obtain counts per million reads (CPM) using TMM (Trimmed Mean of M-values) from the edgeR (v.3.34.1) R package (R v.4.1.0). To control for differences due to DNA sequence 6 , 62 , 74 , 75 , CPM from each experimental sample were divided by CPM from the corresponding naked DNA control. To verify the replicability of the HS damage-seq data, we prepared an extra library from an independent culture of U2OS cells harvested immediately after UV irradiation and sequenced this replicate library to approximately half the depth of the original library.…”