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.
Highlights
The 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
Nanopore libraries Among the two R9 flow cell tested, the first one was run with raw genomic DNA (Library LibRAW) and led to poor results because of the presence of excessive low molecular weight (LMW) fragments (Fig. 3), few active pore numbers, and probably inadequate DNA concentration of the sequencing library
In spite of early difficulties with library preparation due to LMW fragments, we successfully increased read size distribution with minimal equipment using two simple decontamination methods to generate read numbers and yields closer to those previously published for R9 flow cells
Summary
The 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. Two main classes of nanopore reads can be generated - they include 1D reads (D for Dimension or Direction) and higher quality consensus reads formed by reading both the template and complement strands of a given dsDNA molecule. To produce such consensus reads, different adapters are required so that the template and complement can thread successively through the nanopore. This was achieved with a ligated hairpin that physically linked complementary strands into a 2D molecule (up until 2016), while in the latest chemistry (R9.5, released in 2017), linear adapters exhibiting molecular affinity encourage the complementary strand to immediately follow the template strand in the nanopore without direct physical linking, the renaming of 2D to 1D2
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