Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction

Abstract

We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US $5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial.

Novel application of metagenomics for the strain-level detection of bacterial contaminants within non-sterile industrial products – a retrospective, real-time analysis

“…The current workflow does not make this distinction, which would be necessary in cases where background DNA of a related microorganism is abundant. An ideal workflow should also consider emerging cost-saving methodologies [48] to make routine implementation feasible. If the cost could be reduced, then regular surveillance of production facilities could be considered by manufacturers.…”

Genomic Analysis of Carbapenem-Resistant Acinetobacter baumannii Strains Recovered from Chilean Hospitals Reveals Lineages Specific to South America and Multiple Routes for Acquisition of Antibiotic Resistance Genes

“…One milliliter of the liquid culture was used for DNA extraction using the Qiagen DNeasy blood and tissue kit according to the manufacturer’s instructions. Library preparation was performed using the Hackflex protocol ( 32 ), and sequencing was performed on an Illumina MiSeq system at the University of Technology Sydney, Ultimo, sequencing facility. The short reads were assembled using Shovill ( https://github.com/tseemann/shovill ).…”

Plethora of QTLs found inArabidopsis thalianareveals complexity of genetic variation for photosynthesis in dynamic light conditions

“…Genotyping of the DH population was done by extracting DNA via CTAB extraction and consecutively the Hackflex protocol was used to do the library preparation (Gaio et al ., 2022). Samples were pooled, and the fraction showing reads of roughly 500 bp in size were selected and sequenced for on average 1X whole genome coverage sequencing with Novogene (UK) Ltd.…”