2.9. Next-generation sequencing of the capture libraries
Illumina library construction. Post-capture PCR products (16 μl) were
enzymatically shared by adding 2 μl 10X Fragmentase Reaction Buffer v2
and 2 μl NEBNext dsDNA Fragmentase. The sheared DNA (22 μl) was
subjected to end repair, 5′ phosphorylation, dA-tailing and Illumina
adaptor ligation using the NEBNext Ultra DNA Library Prep Kit for
Illumina (NEB) as described by the vendor. PCR enrichment of adaptor
ligated DNA was performed using NEBNext Multiplex Oligos (NEB) with
index primers. The thermal profile was 30 s at 98 °C, followed by eight
cycles of 10 s at 98 °C, 75 s at 63 °C and, 5 min at 72 °C. The PCR
products were finally purified using the Agencourt AMPure XP system as
described in the NEB protocol. The quality of the Illumina library was
verified by checking the size distribution on an Agilent Bioanalyzer
using a high-sensitivity DNA chip. The concentration of the Illumina
library was measured by qPCR using the NEBNext Library Quant Kit for
Illumina (NEB). DNA sequencing was performed using Illumina NextSeq
instrument and standard reagents (Illumina).
Next-generation sequencing computational analyses. To check whether the
sequencing and sample preparation went well, we did a quality check on
the raw data using the FastQC tool (version 0.11.5). Low-quality read
trimming, along with adapter clipping, was performed using Trimmomatic
version 0.36. [35] The resulting fastq files from
the trimmomatic output were then mapped against a reference genome
sequence using Bowtie 2 version 2.4.1 (ref. 15). The reference genome
used was E. coli K12 Using SAMtools. version 0.1.19-44428cd (ref. 16),
we filtered the reads to include only those satisfying
mapping quality scores of at least 30 and then sorted the resulting bam
file. Since the probes were made for genes that satisfy the requirements
of the current protocol, we considered these genes as targets. The rest
of the genes, along with the intergenic regions, were considered as
non-targets.
We then used bed tools version 2.24.0 (ref. 17) to estimate the depth of
the regions. The depth of coverage was normalized to the length of the
target to allow for accurate comparison between targets of different
size. Matches between the reads and the LASSO probes were determined by
using BLAT v35x1. [35]