The advantages of using long reads for de novo assembly of Coturnix japonica rDNA

Molecular genetic analysis methods based on various sequencing technologies are actively used to solve a wide range of problems. Next-generation sequencing (NGS) technology is successful in decoding most DNA sequences, but it has serious limitations due to its relatively short read length, making it impossible to assemble tandem repeats, as well as difficulties in passing sequences with a complex secondary structure.

The latter limits the use of NGS to establish the complete ribosomal gene cluster (rDNA) sequence for most species studied, and especially avian with GC-enriched intrinsic transcribed spacers sequences (ITS1 and ITS2).Among birds, complete information on rDNA cluster organization is currently available only for the chicken genome [1]. Recently, together with her French colleagues,   Alina Zhukova was able to annotate not only the ribosomal genes of Gallus gallus domesticus, but also the GC-enrichedsequences of both internal transcribed spacers (ITS1 and ITS2), as well as the external transcribed spacers (ETS1 and ETS2), and the intergenic spacer (IGS) containing tandem repeats [2].

For the Japanese quail Coturnix japonica there are no such resources, so Alina Zhukova decided to use nanopore sequencing to assemble the entire rDNA de novo for this species. Total genomic DNA was isolated from erythrocyte nuclei of Japanese quail using standard phenol extraction procedures. The quality of the samples was evaluated by capillary electrophoresis using a Qsep device (BiOptic). DNA was quantified using a fluorimeterQubit 4 (Thermo Fisher Scientific) and the recommended reagent kit. To prepare the libraries, NEBNext reagents (New England Biolabs) were used with subsequent sequencing on the MinION device (Oxford Nanopore Technologies).

Long-read nanopore sequencing offers read-through the complex secondary structure of the Japanese quail nucleolar organizer obtaining sequences of about 20kp, covering ETS1, 18S (1860 bp), ITS1, 5.8 S (159 bp), ITS2, 28S (4527 bp), ETS2 and partly IGS.

Alina Zhukova found that, like in the chicken, ITS1 and ITS2 are enriched in GC, which determined the difficulty of their study using polymerase-based sequencing.It was also found that theETS and IGS regions contain long bendable polypyrimidine (polypurine) tracts, which may be responsible for the formation of a complicated secondary structure of these sequences, including the transition to Z-DNA.

1. DyominA.G. et al. 2016. PLoS ONE, 11(6): e0157464. DOI: 10.1371/journal.pone.0157464

2. Dyomin A.G. et al. 2019. Genetics Selection Evolution, 51(59): DOI: 10.1186/s12711-019-0501-7."