Genomic DNA from the parent B. subtilis ATCC 47096 and three JBIR-100-resistant mutants was isolated using an UltraClean Microbial DNA Isolation Kit (MO BIO). Shotgun genomic libraries were constructed and sequenced by the Roy J. Carver Biotechnology Center (University of Illinois, Urbana, IL, USA). The genomic libraries were prepared using a KAPA Hyper Prep Kit (Kapa Biosystems), with average gDNA fragment of 400 bp (from 80 bp to 700 bp). The libraries were quantified by qPCR, and sequenced on one lane on an Illumina HiSeq2500 with single-reads 100 nt in length using a HiSeq SBS sequencing kit version 4. Fastq files were generated and demultiplexed with the bcl2fastq v1.8.4 Conversion Software (Illumina). The parent genome was assembled relative to a reference genome (AL009126.3) using both CLC Genomics Workbench (CLC bio) and Geneious (Biomatters). Polymorphisms in the genomes of the JBIR-100-resistant mutants (P10A, P10B and P10C) were mapped relative to the parent genome and verified by PCR amplification followed by Sanger sequencing.
Hiseq sbs sequencing kit version 4
Manufactured by Illumina
The HiSeq SBS Sequencing Kit Version 4 is a set of reagents used for DNA sequencing on the Illumina HiSeq platform. The kit provides the necessary components for the sequencing-by-synthesis (SBS) method, which is the core technology behind the HiSeq system.
Automatically generated - may contain errors
2 protocols using hiseq sbs sequencing kit version 4
1
Genomic analysis of JBIR-100-resistant B. subtilis
2
NGS Adaptor Addition to Plasmid Libraries
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NGS adaptors were added to the extracted plasmid libraries using the Nextera Index Kit (Illumina, San Diego, CA, USA) with a two-step PCR approach. The first step PCR added the Illumina overhang adapter sequences to all guide sequences (Supplementary Table 12 ) using primers AID-NGS-F1 and AID-NGS-R1. The second step PCR attached Nextera indexes to each library, and the resultant products were gel-purified and quantitated with Qubit (ThermoFisher). ~60 ng of each library was pooled, followed by quantitation by qPCR and sequencing on one lane for 161 cycles from one end of the fragments on a HiSeq 2500 using a HiSeq SBS Sequencing Kit Version 4 (Illumina).
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