Enhancing microbial genome assemblies.

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease (American trypanosomiasis), which affects 6–7 million people worldwide¹. While the majority of infections result in mild symptoms such as fever and headaches, the disease enters a chronic phase where up to 40% of infected individuals may display more severe symptoms (e.g. heart, neurological, and digestive complications), which can manifest 10–30 years after initial infection.

Due to highly repetitive genome sequence, which includes 195 bp satellites, tandem repeats, and multigene families, current short-read based T. cruzi reference genomes are highly fragmented. To address this challenge and enable more accurate comparative genomic studies, Díaz-Viraqué et al.² assessed the utility of long nanopore sequencing reads to generate a more complete genome assembly of this insect-borne parasite.

Using the original Berenice T. cruzi strain first identified by Carlos Chagas in 1909, the team performed whole genome sequencing using both a traditional short-read sequencing platform and the MinION from Oxford Nanopore, which is capable of delivering long sequencing reads.

The short-read assembly alone resulted in 46,821 contigs; however, addition of the nanopore sequencing reads resulted in a 51-fold reduction in the number of contigs (Table 1). Furthermore, the facility of nanopore sequencing to span complex genomic regions resolved an additional 16 Mb of the genome, increasing the assembly size from 25 Mb to 41 Mb.

Table 1: Statistics for short-read only and hybrid (shortand long-read) genome assembly of T. cruzi strain Berenice. Data from Díaz-Viraqué et al.²

Highlighting the superior genome coverage provided by nanopore sequencing reads, the team identified just 54 coverage gaps when aligning the nanopore reads to the hybrid assembly, compared to 3,624 coverage gaps when aligning the short-read data set (Figure 1).

Figure 1: Nanopore sequencing improves T. cruzi assembly contiguity. Example region of T. cruzi genome showing superior coverage of long sequencing reads when compared to short sequencing reads. Figure courtesy of Florence Díaz-Viraqué, Institut Pasteur de Montevideo, Uruguay

‘…we demonstrate that a single run using the MinION sequencer based on a straightforward 10-min library preparation protocol allows a 67-fold increase in genome contiguity and improves genome completeness by 28%...’ ²

The addition of long nanopore sequencing reads also resulted in the increased recovery of protein coding regions, non-coding RNA genes, and transposable elements — supporting the facility to perform functional and comparative studies (Table 2).

Table 2: Long sequencing reads enabled more comprehensive characterisation of the T. cruzi genome than possible using short sequencing reads alone. Data from Díaz-Viraqué et al.²

Confirming the ease of use and cost-effectiveness of nanopore technology, library preparation was performed in just 10 minutes using the Rapid Library Preparation Kit, and all data was obtained using a single MinION Flow Cell. According to the researchers, their approach ‘can be carried out in every laboratory without any previous training in sequencing, contributing to facilitate the enlargement of genomic resources for protozoan pathogens’ ².