|   Application Note, Article

Improving microbial and crude sample bacterial whole-genome sequencing

Fast turnaround times and improved data quality with a single-tube enzymatic fragmentation and library construction method.

Whole-genome sequencing (WGS) of microbes through next-generation sequencing (NGS) technologies can have a major impact on diagnostic microbiology and public health. It enables pathogen identification, differentiation and surveillance on an unprecedented scale and level of resolution.

A greater sequencing capacity, faster sequencing technologies and lower per-genome costs have many benefits, but they demand rapid and robust NGS library construction workflows to support both de novo and re-sequencing applications.

 

Limitations in current methods

Current library construction methods that employ non-mechanical solutions for DNA fragmentation display three key limitations. First, they offer poor control over fragment length, relating to sensitivity with respect to DNA input. Second, there is low library construction efficiency, and third, sequence biases are introduced during fragmentation and/or compulsory library amplification.

The KAPA HyperPlus workflow overcomes these issues and has a number of other benefits. It offers a versatile kit for the construction of DNA libraries for sequencing, and the rapid, one-tube protocol is fully automatable and robust across a wide range of genome GC contents. It also offers flexibility with respect to the amount of input DNA, library fragment size, adapter design, barcoding strategy, and library amplification – while supporting PCR-free workflows.

The combination of a novel, low-bias fragmentation reagent, highly efficient library construction chemistry, and a low-bias amplification enzyme yields high and uniform coverage, thereby facilitating de novo assembly and maximising sequencing cost.

 

Optimised protocols for bacterial WGS

On top of that, KAPA HyperPlus is the only method for WGS that can start from crude lysates. Whole-genome sequencing of bacterial isolates typically involves time-consuming and laborious steps, including bacterial cell culture and the purification of genome DNA (gDNA). The sequencing workflow can be streamlined significantly by a crude DNA extraction protocol, followed directly by library construction. To enable this process, the library construction method must be robust enough to tolerate inhibitors present in crude lysates.

 

Tried and tested

KAPA HyperPlus offers a high-performance, complete and novel workflow solutions for both microbial and crude sample bacterial whole-genome sequencing. The method has been tested on multiple occasions and in different settings, two of which are highlighted here.

The first experiment involved sequencing the genomic DNA of three bacteria from whole genome shotgun libraries, prepared using four different fast library construction strategies. The four methods were compared with respect to key library construction, sequencing, and de novo assembly metrics.

The second study demonstrates the utility of the KAPA HyperPlus Kit for the preparation of gDNA libraries directly from single-colony crude cell lysates of both gram-negative and gram-positive bacteria, bypassing the need for liquid culture and DNA purification. Sequencing and data analysis confirmed that the resulting genome assemblies are comparable to those generated from purified bacterial gDNA.1,2

Would you like to read the reports of these studies? You can download them here.  

For research use only. Not for use in diagnostics procedures.

References

1. Köser, Claudio U., et al., “Rapid single-colony whole genome sequencing of bacterial pathogens.”  Journal of Antimicrobial – Chemotherapy 69.5 (2014): 1275 – 1281.

2. Wright, Meredith S., et al., “SISPA-Seq for rapid whole genome surveys of bacterial isolates.” Infection, Genetics and Evolution – 32 (2015): 191 – 198.