Kapa dna library quantification kit

16S rDNA libraries were quantified with KAPA DNA Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA). Reactions prepared according to the manufacturer’s protocol were performed on LightCycler 480 System (Roche Applied Science, Penzberg, Upper Bavaria, Germany). The libraries were mixed in equimolar ratio and diluted to obtain one 6 pM sequencing library with 10% PhiX Control DNA (Illumina, San Diego, CA, USA). Sequencing was performed on Illumina MiSeq using MiSeq Reagent Kit v3 (600 cycles) (Illumina, San Diego, CA, USA). HPLC-purified custom sequencing primers were mixed with Illumina ones. Demultiplexing of indexed reads was made according to Illumina standard protocol.

The bait design, library preparation, HPV enrichment, and deep sequencing followed methods in original publication [17 (link)]. Briefly, the custom RNA bait (Agilent Technologies Inc., Santa Clara, CA) included 23, 941 probes (each 120 bases in length) complementary to one strand of the full-length genomes of 191 HPV genotypes/subtypes and 12 probes complementary to human haemoglobin subunit beta (HBB). Individual libraries with indexing for sample identification were prepared for each sample. Following indexing, equal amounts of 16 libraries were pooled for enrichment by overnight hybridization to the RNA custom bait and the captured fragments were amplified using 14 PCR cycles. The quality and quantity of HPV-enriched, pooled libraries were assessed by Bioanalyzer 2100 (Agilent Technologies, Inc.) and quantitative PCR using KAPA DNA library quantification kit (KAPA Biosystems, Wilmington, MA) on a LightCycler 480 (Roche Diagnostics, Indianapolis, IN). Each pooled library was paired-end sequenced on an Illumina HiSeq 2500 using TruSeq Rapid SBS Kit HS (200 cycle) (Illumina, San Diego, CA).

Total DNA of PCC6803 and tolerant strains were extracted using the Genomic DNA Buffer Set and QIAGEN Genomic-tips 100/G (Qiagen, CA, USA). A DNA library was prepared using Truseq DNA PCR-Free LT sample prep kit (Illumina, CA, USA) and KAPA DNA library quantification kit (KK4824, KAPA Biosystems, MA, USA), and the sample was sequenced using MiSeq sequencer with MiSeq Reagent kit v2 (Illumina) generating 150 bp paired-end reads. The sequence data were mapped to the genome sequence of PCC6803 GT-I strain (NCBI Reference Sequence No.: NC_017038.1)^{27 (link)} using Bowtie 2 software ver. 2.2.3 with default parameters^{41 (link)}. SNPs were identified using SAMtools ver 1.0 and BCFtools ver. 1.0^{42 (link)} with a threshold of a quality score of >150. The sequence data obtained from MiSeq was deposited in DDBJ Sequence Read Archive under accession number DRA010198. The mutations identified by whole-genome sequencing were confirmed by Sanger sequencing using DNA fragments, including the mutation site amplified by PCR using the primer pair listed in Supplementary Table 4.