title: NextSeq 500 sequencing; LoRTIS: A long read method for analysis of large transposon mutant libraries
design: LoRTIS: A long read method for analysis of large transposon mutant libraries
library_construction_protocol: The Escherichia coli strain BW25113 large transposon mutant library described by Yasir et al. (2020) was used in this study. Briefly: Transposon Tnp001 is a mini-Tn5 transposon coding for kanamycin resistance (aph(3′)-Ia) that incorporates an outward oriented,IPTG-inducible tac promoter, 3′ to the kanamycin-resistance gene Genomic DNA was extracted using the Quick-DNA Fungal/Bacterial 96 Kit (Zymo Research).  For LoRTIS sequencing library preparation,1 μg of genomic DNA was tagmented using an Illumina compatible MuSeek library preparation kit (Thermo Fisher Scientific) and the tagmented DNA was cleaned using 0.5X volume of AMPure XP magnetic beads (Beckman-Coulter).  LongAmp Taq DNA polymerase (New England Biolabs, UK) was used with biotinylated oligonucleotide primers that hybridise specifically within the transposon to amplify DNA fragments containing transposon insertion junctions. A second primer that anneals to MuSeek adapters ('IonTMu-02') was used to generate biotinylated double-stranded PCR products containing transposon-genome junctions. These PCR products were enriched using a Dynabeads™ kilobaseBINDER™ kit (Thermo Fisher Scientific).  A nested PCR reaction was carried out using NEB LongAmp Taq polymerase to generate DNA fragments with Oxford Nanopore multiplexing adapters that lacked biotin.