Lagging strand detection with SDL-TOPO protocol

SDL-TOPO protocol was applied for the detection of lagging-strand in replicating budding yeast strain. Genomic DNA was extracted from cdc9 mutant strain, and served for library preparation with SDL-TOPO. To see the sensitivity of SDL-TOPO for the detection of lagging strand, various amounts of genomic DNA were used for the library preparation: 75 ng, 25 ng, 7.5 ng, 2.5 ng, 750 pg, 250 pg, 75 pg, and 25 pg. The prepared libraries were sequenced on Illumina iSeq100 with paired-end mode of 150x2. Only read 1 was served for mapping with bowtie2 against reference sequence sacSer3. The mapped reads were separated strand specifically, and read depths were visualized. The strand-biased mapping patterens were observed for libraries prepared from each amount of input DNA. To detect lagging strand DNA fragment, cdc9 mutant cells were used. Genomic DNA extracted from the strain was firstly denatured, and served for library preparation with SDL-TOPO protocol. Since SDL-TOPO can generate sequencing library from single-stranded DNA, any types of DNA can be converted to sequencing library. Because leading strand and template genomic DNA are too long to be amplified with indexing PCR, only library molecules originated from lagging strand can be amplified and indexed.