De-novo Assembly of Limnospira fusiformis Using Ultra-Long Reads

McKenna Hicks,Thuy-Khanh Tran-Dao,Logan Mulroney,David L. Bernick

doi:10.3389/fmicb.2021.657995

McKenna Hicks, Thuy-Khanh Tran-Dao + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2021.657995

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Abstract

The Limnospira genus is a recently established clade that is economically important due to its worldwide use in biotechnology and agriculture. This genus includes organisms that were reclassified from Arthrospira, which are commercially marketed as “Spirulina.” Limnospira are photoautotrophic organisms that are widely used for research in nutrition, medicine, bioremediation, and biomanufacturing. Despite its widespread use, there is no closed genome for the Limnospira genus, and no reference genome for the type strain, Limnospira fusiformis. In this work, the L. fusiformis genome was sequenced using Oxford Nanopore Technologies MinION and assembled using only ultra-long reads (>35 kb). This assembly was polished with Illumina MiSeq reads sourced from an axenic L. fusiformis culture; axenicity was verified via microscopy and rDNA analysis. Ultra-long read sequencing resulted in a 6.42 Mb closed genome assembled as a single contig with no plasmid. Phylogenetic analysis placed L. fusiformis in the Limnospira clade; some Arthrospira were also placed in this clade, suggesting a misclassification of these strains. This work provides a fully closed and accurate reference genome for the economically important type strain, L. fusiformis. We also present a rapid axenicity method to isolate L. fusiformis. These contributions enable future biotechnological development of L. fusiformis by way of genetic engineering.

Highlights

Limnospira is a recently established genus that contains three species reclassified from Arthrospira based on morphological, molecular, and ecological differences between the two genera (NowickaKrawczyk et al, 2019)
We performed nanopore sequencing on DNA that was extracted from a physically treated culture that resulted in predominantly L. fusiformis DNA (Figure 2B and Supplementary Figure 13)
Long-read sequencing enabled closure of the Limnospira fusiformis genome in a single pass

Summary

Introduction

Limnospira is a recently established genus that contains three species reclassified from Arthrospira based on morphological, molecular, and ecological differences between the two genera (NowickaKrawczyk et al, 2019). Their cultivation is simple, inexpensive, and their alkaline growth preference makes commercial production less prone to living contaminants (Kebede and Ahlgren, 1996; Nowicka-Krawczyk et al, 2019) These aspects make Limnospira/Arthrospira attractive for use in many biotechnological and agricultural applications, including wastewater treatment, biofuels, biomanufacturing, medicine, Limnospira fusiformis Complete Genome Assembly and nutritional supplementation (Belay et al, 1993; Tokusoglu and üUnal, 2003; Belay and Gershwin, 2007; Abed et al, 2016; Jiang et al, 2017; Aikawa et al, 2018; Czerwonka et al, 2018; Mehan et al, 2018; Zinicovscaia et al, 2018). Known as “Spirulina” in commercial settings (Sili et al, 2012; NowickaKrawczyk et al, 2019)

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