Hackflex: low cost Illumina Nextera Flex sequencing library construction

Abstract

We developed Hackflex, a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 11 times more libraries for high-throughput Illumina sequencing to be generated at a fixed cost. We call this new method Hackflex. Quality of library preparation was tested by constructing libraries from E. coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. We demonstrated that Hackflex can produce high quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. Using Hackflex, we were able to achieve a per sample reagent cost of library prep of A$8.66, which is 8.23 times lower than the Standard Nextera Flex protocol at advertised retail price. An additional simple modification to the protocol enables a further price reduction of up to 11 fold or about A$6.50/sample. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programs where sequencing large numbers of libraries is beneficial .

Community composition and development of the post-weaning piglet gut microbiome

“…Sample index barcode design using a previously introduced method 105 yielded a set of 96 × 8nt sequences with a 0.5 mean GC content and none of the barcodes containing 3 or more identical bases in a row. Nine hundred-sixty different combinations of i5 and i7 primers were used to create a uniquely barcoded library for each sample.…”

“…The detailed sample-to-barcode assignment is given in Supplementary le 3. Library preparation was carried out using a modi cation of the Nextera Flex protocol to produce low bias, low cost shotgun libraries, as described in a previous manuscript 105 . Following the ampli cation step, samples were centrifuged at 280 x g for 1 min and stored between 1 and 5 days at 4 °C.…”

“…Forty microliters from each of the 10 plate pools and 40 µL from the master pool underwent library size selection and puri cation using equal volumes of SPRIselect beads (Beckman Coulter) and ultrapure water (Invitrogen). Sample cleaning with SPRI-beads was performed as described previously 105 . A puri ed master pool comprising samples from all plates, and puri ed pools of individual plates to check for plate-speci c anomalies, were diluted to 4 nM and fragment size distribution was assessed using the High Sensitivity DNA kit on the Bioanalyzer (Agilent Technologies, USA).…”

“…were performed. Alpha-diversity and beta-diversity were analyzed and the results were visualized with R 108 and R packages [91][92][93][94][95][96][97][98][99][100][101][102][103][104][105][106][107] . The data analysis is schematically represented in Supplementary Fig.…”

Optimized and affordable high‐throughput sequencing workflow for preserved and nonpreserved small zooplankton specimens

“…Dedicated kits such as NEBNext Ultra II FS can be viable alternatives to the modified Nextera Kit, as its low‐cost advantage (Baym et al., 2015; Therkildsen & Palumbi, 2017) diminishes when the amount of input DNA is increased due to more of the relatively expensive transposome being required. While, the Nextera kit is no longer manufactured, there are new protocols available for modifying the current Nextera DNA Flex Kit from Illumina (Gaio et al., 2019), which can be modified in a similar way to the changes proposed here to reduce DNA input below the suggested 10 ng.…”