Femto pulse

A SMRT bell library was produced using the SMRTbell Express Template Prep Kit (Pacific Biosciences 101-357-000). 10 μg of gDNA were mechanically sheared to an average size distribution of 15-20 kb, using a Covaris gTube (Covaris p/n 520079). 3 μg of sheared gDNA was DNA damage repaired and end-repaired using polishing enzymes. A ligation reaction was performed to create the SMRT bell template, according to the manufacturer’s instructions. A Blue Pippin device (Sage Science) was used to size select the SMRT bell template and enrich the big fragments beyond 10 kb. The sized selected library was quality inspected and quantified using a Femto Pulse (Agilent) gDNA analysis assay and on a Qubit Fluorometer respectively. A ready to sequence SMRT bell-Polymerase Complex was created using the P6 DNA/Polymerase binding kit 2.0 (Pacific Biosciences p/n 100-236-500) according to the manufacturer instructions. The Pacific Biosciences RSII instrument was programmed to load and sequence the sample on 1 SMRT cell v3.0 (Pacific Biosciences p/n100-171-800), taking 1 movie of 360 minutes. A MagBead loading (PacBio p/n 100-133-600) method was chosen in order to improve the enrichment of the longer fragments.

All reagents are described in Supplementary Data 7. DNA was extracted from 200 RevI-H2i2 females using the Qiagen Blood & Cell Culture DNA Midi kit. The genomic DNA quality and quantity were evaluated using a Femto Pulse (Agilent) and a Qbit 3.0 (Invitrogen) respectively. Oxford Nanopore Technology (ONT) sequencing was performed by I2BC (Gif-sur-Yvette, France) using five micrograms of genomic DNA. Adapter-trimmed ONT reads were analyzed using NCBI Blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) or command-line Blastn (Blastn 2.8.1). The RevI-H2i2 ONT reads containing ZAM insertions were detected by Blastn with the reference ZAM^{22 (link)}, Repbase ZAM_I and ZAM_LTR sequences (https://www.girinst.org/repbase/^{31 (link)},). The ZAM containing reads were then recovered with bedtools getfasta (https://bedtools.readthedocs.io/en/latest/content/tools/getfasta.html^{70 (link)},). The ZAM insertion sites where determined using Blastn of the ZAM containing reads to the D. melanogaster Release 6 genome (http://flybase.org). The absence of empty sites, without a given ZAM insertion, was verified by Blastn of the respective empty insertion sites, recovered from the D. melanogaster Release 6 genome, to all ONT reads. Sequencing data are available in NCBI GEO database, accession GSE213456.

Three biomass samples were taken at weeks 1, 20, and 40 from a year long sampling campaign, directly from an AD reactor digesting food waste by the facility operators and shipped in ice-cooled containers to the University of Warwick. Upon receipt, they were stored at 4°C and then sampled into several 1–5mL aliquots within a few days and stored in 1.8 mL Cryovials at −80°C. Samples were defrosted at 4°C overnight prior to DNA extraction. DNA was extracted from a starting mass of 250 mg of anaerobic digester sludge using the MP Biomedical FastDNA SPIN Kit for Soil (cat no: 116560200) and a modified manufacturers protocol (see [27 (link)] for detailed protocol).
DNA size was assessed using a FemtoPulse (Agilent). The Pacific Biosciences protocol ‘Preparing 10 kb Library Using SMRTbell R Express Template Prep Kit 2.0 for Metagenomics Shotgun Sequencing’ was used to create libraries from 1.5 micrograms of DNA. In most cases the DNA was already 10 kb or smaller. Sample AD2W40 was a bit larger so the DNA was sheared using a g-TUBE (Covaris) for one library and unsheared for a second library. Libraries were not pooled due to the large number of reads desired. Sequencing was performed using a Sequel II sequencer (Pacific Biosciences) using version 8M SMRT cells and version 2.0 sequencing reagents with 30-hour movies with 2 hr pre-extension time to generate CCS reads.

For long-read PacBio sequencing, high-molecular weight (HMW) DNA of C. japonica was isolated from root cultures using the NucleoBond HMW DNA kit (Macherey Nagel, Germany), quality was assessed with a FEMTOpulse device (Agilent, USA), and quantity was measured by the Quantus fluorometer (Promega, USA). A HiFi library was then prepared according to the “Procedure & Checklist - Preparing HiFi SMRTbell® Libraries using SMRTbell Express Template Prep Kit 2.0” manual with an initial DNA fragmentation by Megaruptor 3 (Diagenode, Belgium) and final library size binning into defined fractions by SageELF (Sage Science, USA). Size distribution was again controlled by FEMTOpulse (Agilent, USA). Polymerase-bound SMRTbell complexes were formed according to standard protocols (Pacific Biosciences of California Inc., USA) and loaded at an on-plate concentration of 85 pM (14, 15, 20, and 26 kb mean length). SMRT sequencing was performed using one 8 M SMRT cell per library (30 h movie time, 2 h pre-extension time) on the Pacific Biosciences Sequel II device, generating a total of 80 Gb (HiFi CCS). The SMRTbell libraries were sequenced at IPK Gatersleben.

For haplotype-phased genome assembly, a wild individual kept in an aquaculture farm at Ainoshima Island, Fukuoka, Japan (hereafter we refer this individual as to AI) was used. For the haplotype-merged genome assembly, we obtained an inbred individual produced through three successive sib-mating generations at the Pearl Research Institute of K. MIKIMOTO & CO., LTD, Shima, Japan (MK). The experimental procedure and methods for the haplotype-merged MK assembly construction are described in Supplementary Text.
For specimen AI, an adductor muscle was sampled, immediately frozen in liquid nitrogen, and stored at −80°C until DNA extraction. High-molecular-weight genomic DNA was extracted using a Bionano Prep Blood and Cell Culture DNA isolation Kit (Bionano Genomics, CA, USA) following the manufacturer’s instructions. The size distribution and concentration of the extracted DNA were assessed using a FEMTO Pulse (Agilent Technologies, CA, USA) and a Qubit Fluorometer (Thermo Fisher Scientific, MA, USA) devices.

Three biomass samples were taken at weeks 1, 20, and 40 from a year long sampling campaign, directly from an AD reactor digesting food waste by the facility operators and shipped in ice-cooled containers to the University of Warwick. Upon receipt, they were stored at 4°C and then sampled into several 1–5mL aliquots within a few days and stored in 1.8 mL Cryovials at −80°C. Samples were defrosted at 4°C overnight prior to DNA extraction. DNA was extracted from a starting mass of 250 mg of anaerobic digester sludge using the MP Biomedical FastDNA SPIN Kit for Soil (cat no: 116560200) and a modified manufacturers protocol (see [27 (link)] for detailed protocol).
DNA size was assessed using a FemtoPulse (Agilent). The Pacific Biosciences protocol ‘Preparing 10 kb Library Using SMRTbell R Express Template Prep Kit 2.0 for Metagenomics Shotgun Sequencing’ was used to create libraries from 1.5 micrograms of DNA. In most cases the DNA was already 10 kb or smaller. Sample AD2W40 was a bit larger so the DNA was sheared using a g-TUBE (Covaris) for one library and unsheared for a second library. Libraries were not pooled due to the large number of reads desired. Sequencing was performed using a Sequel II sequencer (Pacific Biosciences) using version 8M SMRT cells and version 2.0 sequencing reagents with 30-hour movies with 2 hr pre-extension time to generate CCS reads.

PacBio HiFi data were generated from the HG00733 lymphoblastoid cell line as previously described (G. A. Logsdon et al., 2021 (link)) with modifications. Briefly, DNA was extracted from 4.3×10^6 cells using the Monarch HMW DNA Extraction Kit for Cells and Blood (New England Biolabs) with 1400 rpm lysis speed. After UV absorption and fluorometric quantification (Qubit High Sensitivity DNA kit, Thermo Fisher) on the DS-11 FX instrument (Denovix) and evaluation of DNA integrity on FEMTO Pulse (Agilent), 12 μg of DNA was prepared for sequencing using Megaruptor 3 shearing (Diagenode, settings 19/31) and the Express Template Prep Kit v2 and SMRTbell Cleanup Kit v2 (PacBio). The library was size-selected on a PippinHT instrument (Sage Science) using a 15 kbp high-pass cut. Five SMRT Cell 8Ms were run on a Sequel II instrument using Sequel II chemistry C2.0/P2.2 with 30-hour movie times, 2-hour pre-extension, and adaptive loading targets of 0.8–0.85 (PacBio). Circular consensus calling was performed with CCS version 6.0.0 (SMRT Link v.10.1) and reads with estimated quality scores ≥Q20 were selected for downstream analysis.