Nebnext ultra dna library prep kit for sequencing

Genomic DNA of E. coli TolC was extracted by gravity flow using the Genomic-tip 20/G kit (Qiagen, Germany). Purified genomic DNA of E. coli TolC was processed for PacBio SMRT sequencing and Illumina MiSeq paired-end sequencing (2 × 250 bp) with a target genome coverage of 150-fold. DNA libraries for MiSeq sequencing of the genome of E. coli TolC were prepared with the NEBNext Ultra DNA library prep kit for Illumina sequencing (New England Biolabs, Ipswich, MA). Quality controls of NEBNext Ultra DNA libraries were conducted by fluorometric quantitation using the Qubit 3.0 fluorometer (Thermo, Fisher Scientific, Germany). For PacBio SMRT sequencing, a PacBio SMRTbell library was constructed according to the manufacturer’s instructions and the library was sequenced on the PacBio RSII platform. De novo genome assemblies were built with PacBio’s SMRT Portal (v.2.3.0) by utilizing the Hierarchical Genome Assembly Process 3 (HGAP3) (46 (link)). The genome was error corrected against indel errors by a mapping of Illumina reads onto finished genomes, using BWA (47 (link)) with subsequent variant and consensus calling using VarScan (48 (link)); automated sequence annotation was performed with Prokka (v.1.8) (49 (link)).

16S rRNA sequencing was performed following the method described by our group (Mukherjee et al., 2014 (link), 2016 (link)) with minor modifications. In brief, library preparation was performed using the NEBNext Ultra™ DNA Library Prep Kit for Illumina sequencing (New England Biolabs), following the manufacturer’s recommendations. PCR was performed by thermocycling: 5 min at 94°C for initialization; 28 cycles of 3 min denaturation at 94°C, 40 s annealing at 53°C, and 1 min extension at 72°C; followed by 5 min final elongation at 72°C. We used three replicates per sample, and each PCR product of the same sample was mixed. The amplicon products from different samples were purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, Beverly, MA, USA). The library quality was assessed on a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2,100 system. Finally, the library was sequenced on an Illumina Hiseq 2,500 platform, and 250-bp paired-end reads were generated.

Two genomic DNA pools, one for high acidity (12.6 ± 2.5 mg ml⁻¹) and the other for regular acidity (5.7 ± 0.6 mg ml⁻¹), were created using 18 and 20 progenies from the GMAL 4595 population, respectively (Table S2, Fig. 1c). Since Ma3 was a non-factor in this population and the progenies in the high-acidity pool comprising five MaMa and 13 Mama progenies, similar to those in the regular acidity pool, i.e., 5 in MaMa and 15 in Mama, the acidity differences between the two pools were assumed to be caused by genetic factors other than Ma and Ma3. Genomic DNA samples were extracted from young leaves, and the DNA samples were assessed on 1% agarose gel for integrity, and were quantified with a Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). For each pool, an equal amount of DNA (1 µg) from each progeny was combined to produce the pooled genomes (Fig. 1c). Construction of the pooled genomic libraries was conducted using NEBNEXT Ultra DNA library prep kit for Illumina sequencing (New England Biolabs, E7370) with a targeted insert size of 500 bp. The libraries were sequenced in paired-end (2 × 151 bp) on an Illumina NextSeq 500 platform (Fig. 1d) at the Genomics Facility of Cornell University (Ithaca, NY, USA).

Sequencing libraries were generated using the NEBNext^® Ultra^TM DNA Library Prep Kit for Illumina^® sequencing (New England Biolabs, United States) following the manufacturer’s recommendations, and index codes were added. The library quality was assessed on a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina Hiseq2500 platform, and 250 bp paired-end reads were generated.