DOI: 10.5061/dryad.2fqz612t6
title: Chromosome-scale thraustochytrid genome assembly

description: We used long-read sequencing to produce a telomere-to-telomere genome
 assembly for the osmoheterotrophic stramenopile protist Aurantiochytrium
 limacinum MYA-1381. Its ~62 Mb genome is mainly organized in 26 linear
 chromosomes with a novel configuration: subtelomeric rDNAs are
 interspersed with long repeated sequence elements denoted as LOng REpeated
 - TElomere And Rdna Spacers (LORE-TEARS). These repeats may play a role in
 chromosome end maintenance. A putative circular mirusvirus genome is
 present at a high copy number (called circular element 1; CE1). The
 presence of another mirusvirus genome at the end of chromosome 15 in
 superposition between two complete sets of rRNA and LORE-TEAR elements
 suggests a dynamic process at the chromosome ends.
description: For Nanopore sequencing, Aurantiochytrium
 ATCC MYA-1381 and two putative crtIBY knockout mutants (designated KO32
 and KO33; (Rius et al. 2023)) were cultured for three days in 50 ml ATCC
 790 By+ medium. Genomic DNA was extracted as described in the manuscript.
 The precipitated DNA was left to dissolve in water by spontaneous
 diffusion for 48+ hours at room temperature to avoid shearing and
 subsequently purified using QIAGEN Genomic-tip 20/G. Agarose gel
 electrophoresis (1%) was used to visually assess and confirm the integrity
 of high molecular weight (20+ Kb) DNA. DNA quality was evaluated using a
 NanoPhotometer P360 (Implen) to measure A260/280 (~1.8) and A260/230
 (2.0-2.2) ratios. The quantity of DNA was calculated using a Qubit 2.0
 Fluorometer (ThermoFisher Scientific) with the dsDNA broad range assay
 kit. A multiplexed Nanopore
 (MinION, Oxford Nanopore Technologies) sequencing library was prepared
 using the Oxford Nanopore Technology (ONT) ligation sequencing kit
 (SQK-LSK109) and the PCR-free native barcoding expansion kit 1-12
 (EXP-NBD103) according to the Oxford Nanopore Technologies protocol “1D
 Native barcoding genomic DNA with EXP-NBD103 and SQK-LSK109 (version
 NBE_9065_v109_revB_23May2018). Approximately 2 µg of purified genomic DNA
 per sample were used as input. Unfragmented genomic DNA for the wild-type
 and putative knockouts was repaired using the NEBNext FFPE DNA repair
 module (NEB cat. no. M6630) and prepared for adapter ligation using the
 NEBNext End repair/dA-tailing module (NEB cat. no. E7546) with incubations
 at 20°C and 65°C for 10 minutes each. The DNA repaired/end-prepped samples
 were purified with a 1:1 volume of AMPure XP beads (Beckman), and
 subjected to an incubation at room temperature for 10 minutes; the
 pelleted beads were subsequently washed twice with 80% ethanol. The DNA
 was eluted off the beads in 25 µl nuclease free water for 10 minutes at
 37°C to encourage the elution of long molecules from the beads. The native
 barcodes NB07, NB08, and NB09 were ligated to the WT, KO32, and KO33
 repaired/end-prepped DNA samples, respectively, in a 1.36x scaled ligation
 reaction. Each native barcoded sample was pooled in approximately
 equimolar amounts (~1.3 µg each). The 1D barcode sequencing adapters (BAM
 1D) were then ligated to the pooled and barcoded DNA using a 1-hour
 incubation at 25°C. The adapter ligated DNA was purified by a 0.4x AMPure
 XP bead clean-up including a 10 minute incubation at room temperature and
 two washes using the Long Fragment Buffer mix to enrich for DNA fragments
 &gt;3 Kbp. The final adapter ligated library was incubated in 15 µl
 Elution Buffer for 10 minutes at 37°C. A total of 1.2 µg of prepared
 library was loaded on a single MinION R9.4.1 chemistry SpotON flow cell
 (FLO-MIN106) and sequenced via Oxford Nanopore Technology's MinKNOW
 software (v2.1.12) without live basecalling. Binning of the raw reads was
 performed in real time using Deepbinner v0.2.0 (Wick, Judd, and Holt
 2018)(Rius et al. 2023).The raw fast5 MinION data has been deposited in
 the NCBI SRA database BioProject PRJNA680238 (WT accession: SRR13108467;
 KO32 accession: SRR13108466; KO33 accession: SRR13108465). As described previously (Rius et al. 2023), the
 demultiplexed fast5 files were base called using Albacore v2.3.1, adapters
 were removed by Porechop v0.2.3 (Wick et al. 2017), and the resulting data
 were used for preliminary genome assembly by Canu v1.7.1 (Koren et al.
 2017) with parameters adjusted to the expected genome size of 60 Mbp. The
 resulting consensus sequence was improved by Nanopolish v0.10.1 (Loman,
 Quick, and Simpson 2015) (https://github.com/jts/nanopolish). For
 wild-type, the genome assembly totaled 61.9 Mbp in 55 contigs, while the
 genomes of KO mutants 32 and 33 both assembled as 62.5 Mbp into 50 and 47
 contigs, respectively. Analysis by Mauve v2.4.0 (Darling et al. 2004)
 revealed ~27 homologous contigs among the three assemblies, ranging from
 ~0.35 up to ~4 Mbp. The wild-type assembly was the least contiguous,
 perhaps reflecting its lower mean read length (4913 bp vs 8508 and 7951).
 In an effort to resolve the differences among the three assemblies and
 gain greater coverage, reads for all three strains were concatenated into
 one file as input for Canu v1.7.1, and read trimming and assembly were
 performed with default settings and an estimated genome size of 60 Mb. The
 combined read file had 789085 reads &gt;Q7 and 293668 reads
 &gt;Q10, mean read length 7124 bp and the longest read was 180475 bp
 (quality score 9.8). This combined Nanopore-based Canu assembly contained
 62 contigs and a total of 63.71Mbp. The 27 contigs described in the
 associated manuscript total 61.77 Mbp. The remaining 35 contigs included
 five putative mitochondrial contigs and another 30 contigs ranging from 20
 Kb to 230 Kb, of which 14 represented single reads.  <strong>References</strong>
 Darling, Aaron C. E., Bob Mau,
 Frederick R. Blattner, and Nicole T. Perna. 2004. “Mauve: Multiple
 Alignment of Conserved Genomic Sequence with Rearrangements.” Genome
 Research 14 (7): 1394–1403. Koren, Sergey, Brian P. Walenz, Konstantin Berlin,
 Jason R. Miller, Nicholas H. Bergman, and Adam M. Phillippy. 2017. “Canu:
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 Simpson. 2015. “A Complete Bacterial Genome Assembled de Novo Using Only
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