Promising prospects of nanopore sequencing for algal hologenomics and structural variation discovery

Abstract

BackgroundThe MinION Access Program (MAP, 2014–2016) allowed selected users to test the prospects of long nanopore reads for diverse organisms and applications through the rapid development of improving chemistries. In 2014, faced with a fragmented Illumina assembly for the chloroplast genome of the green algal holobiont Caulerpa ashmeadii, we applied to the MAP to test the prospects of nanopore reads to investigate such intricacies, as well as further explore the hologenome of this species with native and hybrid approaches.ResultsThe chloroplast genome could only be resolved as a circular molecule in nanopore assemblies, which also revealed structural variants (i.e. chloroplast polymorphism or heteroplasmy). Signal and Illumina polishing of nanopore-assembled organelle genomes (chloroplast and mitochondrion) reflected the importance of coverage on final quality and current limitations. In hybrid assembly, our modest nanopore data sets showed encouraging results to improve assembly length, contiguity, repeat content, and binning of the larger nuclear and bacterial genomes. Profiling of the holobiont with nanopore or Illumina data unveiled a dominant Rhodospirillaceae (Alphaproteobacteria) species among six putative endosymbionts. While very fragmented, the cumulative hybrid assembly length of C. ashmeadii’s nuclear genome reached 24.4 Mbp, including 2.1 Mbp in repeat, ranging closely with GenomeScope’s estimate (> 26.3 Mbp, including 4.8 Mbp in repeat).ConclusionOur findings relying on a very modest number of nanopore R9 reads as compared to current output with newer chemistries demonstrate the promising prospects of the technology for the assembly and profiling of an algal hologenome and resolution of structural variation. The discovery of polymorphic ‘chlorotypes’ in C. ashmeadii, most likely mediated by homing endonucleases and/or retrohoming by reverse transcriptases, represents the first report of chloroplast heteroplasmy in the siphonous green algae. Improving contiguity of C. ashmeadii’s nuclear and bacterial genomes will require deeper nanopore sequencing to greatly increase the coverage of these larger genomic compartments.

Identification of polycistronic transcriptional units and non-canonical introns in green algal chloroplasts based on long-read RNA sequencing data

“…1 and Additional file 1 : Tables S1-S3). Analysis of the assembly graph of our long-read DNA sequencing data did not identify any structural heterogeneity, unlike the recent observations for another Caulerpa species [ 18 ]. The differences between the C. lentillifera chloroplast genomes Clcp-v1 and MG753774.1 appear to reflect strain-level variation.…”

“…Since the raw reads for the previously published genome are not available, we cannot determine the exact reason for the differences between the two sequences at present. Long-read sequences also permit identifying heteroplasmy within individuals, as recently shown in the chloroplast genome of a related species by nanopore sequencing [ 18 ]. Our PacBio long-read data did not reveal any evidence of such structural variations, and in our opinion the prevalence and nature of heteroplasmy across the siphonous green algae requires further work based on long-read methods that deliver highly accurate reads.…”

Identification of Polycistronic Transcriptional Units and Non-canonical Introns in Green Algal Chloroplasts Based on Long-read RNA Sequencing Data

“…1 and Additional le 1: Table S1-S3). Analysis of the assembly graph of our long-read DNA sequencing data did not identify any structural heterogeneity, unlike the recent observations for another Caulerpa species [17]. The differences between the C. lentillifera chloroplast genomes Clcp-v1 and MG753774.1 rather appears to re ect strain-level variation.…”

“…Since the raw reads for the previously published genome are not available, we cannot determine the exact reason for the differences between the two sequences at present. Long-read sequences also permit identifying heteroplasmy within individuals, as recently shown in the chloroplast genome of a related species by nanopore sequencing [17]. Our PacBio long-read data did not reveal any evidence of such structural variations, and in our opinion the prevalence and nature of heteroplasmy across the siphonous green algae requires further work based on long-read methods that deliver highly accurate reads.…”

Multiple Intraspecific Variations of Mitochondrial Genomes in the Green-Tide Forming Alga, Ulva compressa Linnaeus (Ulvophyceae, Chlorophyta)

“…The reported ulvophycean mitogenomes exhibit large variation in genome size, architechtures, gene density, and intron content (Liu et al, 2017). Species in Bryopsidales harbor the inflated mitogenomes ranged from 197,427 bp in Caulerpa ashmeadii to 241,739 bp in Ostreobium quekettii, due to expansion of noncoding DNA and proliferation of introns (Sauvage et al, 2019;Repetti et al, 2020). Species in Ulotrichales tend to contain large mitogenomes, such as the 105,236-bp Gloeotilopsis planctonica and 95,880-bp Pseudendoclonium akinetum mitogenomes, which also display characteristics of the 'expanded-derived' patterns of evolution (Pombert et al, 2004;Turmel et al, 2016).…”