X ten

C. enshiensis samples (3 g) were washed twice in cold PBS, crosslinked with 1% formaldehyde for 10 min at room temperature, and then quenched by the addition of glycine (125 mmol/L final concentration). Afterwards, the samples were lysed, and chromatin was obtained on ice. The chromatin samples were sonicated to obtain soluble sheared chromatin (average DNA length of 200–500 bp). Twenty microliters of chromatin was saved at –20 °C as input DNA, and 100 µL of chromatin was used for immunoprecipitation with H3K27me3 antibodies (CST9733, Cell Signaling Technology) and H3K4me2 antibodies (CST9725, Cell Signaling Technology). The immunoprecipitated DNA was used to construct sequencing libraries following the protocol provided by the I NEXTFLEX^® ChIP-Seq Library Prep Kit for Illumina^® Sequencing (NOVA-514120, Bioo Scientific) and sequenced on Illumina X Ten with the 150PE method by Wuhan IGENEBOOK Biotechnology Co., Ltd (http://www.igenebook.com).

Total RNA samples were shipped on dry ice to an affiliation of the Beijing Genome Institute (BGI Tech Solutions, Hong Kong) for library preparation, sequencing, and bioinformatic analysis (coordinated by BGI Tech Solutions, Shenzhen, China). Sample quality and RNA concentrations were checked using the Agilent model 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and approved for sequencing (RNA Integrity Number, RIN: 9.7, and 28S/18S: 1.7). 8.6 microgram RNA was used to construct two cDNA libraries separately. The PacBio Iso-Seq libraries with a size of 0-5 kb (Pacific Biosciences, Menlo Park, CA, USA) were generated for sequencing on two SMRT cells and one RNA-Seq library was prepared for sequencing with Illumina X Ten (Illumina, San Diego, CA, USA).

Library preparation was conducted following Peterson et al. (2012) (link). The genomic DNA (100 ng 10 μl) was double digested using 10 μl of the restriction enzymes EcoR I and Mse I for 5 h at 37°C, then 20 min at 65°C, and final incubation at 12°C. The resulting digested fragments were cleaned and subsequently quantified using agarose gel electrophoresis. Digested fragments were ligated to EcoR I and Mse I adapters containing sample specific barcodes with T4 DNA ligase (NEB) for 4 h at 16°C, then 20 min at 65°C, and final incubation at 12°C. Individually barcoded samples were cleaned and size-selected (350–500 bp) using agarose gel (Omega kit). Each library was then PCR-amplified to the desired concentration and paired-end sequenced (0.5 G each sample) on an Illumina X-ten (Illumina) with PE 150 mode.

Thirteen paired-end libraries (150 bp) with insert sizes of ~270 bp were constructed according to the manufacturer’s instructions. A total of 430 Gb of short reads was obtained for genome survey and polishing. PacBio sequencing libraries were constructed as recommended by Pacific Biosciences. DNA fragments of about 10–50 kb were selected using BluePippin electrophoresis. Next, libraries were constructed and sequenced on the PacBio Sequel system with P6-C4 chemistry. A total of 120 SMRT cells were sequenced, producing 497 Gb of raw data. Hi-C libraries were created using a previously described method^{15 (link)}. Six Hi-C fragment libraries, including five DpnII and one HindIII libraries, with fragment sizes ranging from 300 to 700 bp, were constructed and sequenced on the Illumina X Ten platform. A total of 1,869,066,895 paired reads (560 Gb of raw data) were generated.

These callsets, generated independently for each individual in the Ashkenazi trio, used LongRanger^{21 (link)} (version 2.2, code at https://github.com/10XGenomics/longranger) and GATK v4.0.0.0 as variant caller with default parameters on 10x Genomics linked-reads data for the family trio (84x, 70x, and 69x coverage for HG002 NA24385 son, HG003 NA24149 father, and HG004 NA24143 mother, respectively) against both GRCh37 and GRCh38. The vcf and bam files for each genome are under:
ftp://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/AshkenazimTrio/analysis/10XGenomics_ChromiumGenome_LongRanger2.2_Supernova2.0.1_04122018/The variant curation used the 10x Genomics VCF from LongRanger 2.2 derived from SRA accession SRX2225480 [https://www.ncbi.nlm.nih.gov/sra/SRX2225480], which is available at: ftp://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/AshkenazimTrio/analysis/10XGenomics_ChromiumGenome_LongRanger2.2_Supernova2.0.1_04122018/GRCh37/NA24385_300G/NA24385.GRCh37.phased_variants.vcf.gzAll samples were sequenced on the Illumina Xten at 2 × 150bp. The Ashkenazim trio was done using the v1 of the 10x library prep protocol.

We constructed genomic libraries using the TruSeq DNA Nano kit with a DNA insert size of 350 bp. Sequencing was conducted on the Illumina X Ten platform, which generated at least 21 Gb raw data from each species. Sequence data were quality trimmed using SOAPnuke (with the options: -n 0.1 -| 20 –q 0.1 -5 1) (Chen et al., 2018 (link)). Sequences were assembled into contigs according to the protocol described by Hahn, Bachmann & Chevreux (2013) (link) using MIRA sequence assembler software (default settings) with the reference genome I. trifida (accession number: KF242476.1). The reads were blasted into contigs with perl scripts and contigs were extended to obtain the complete sequence. Finally, we blasted the reads to the complete sequence, detected the coverage of reads and manually corrected the sequence. The services of library construction, sequencing, and assembly were provided by Macrogen (http://www.macrogencn.com/sy, Shenzhen, China).
The eight chloroplast genome sequences were initially annotated using the online CpGAVAS (Liu et al., 2012 (link)) software with default settings, and then manually corrected using Genious 11.0.5. The circular chloroplast genome maps were constructed using the OrganellarGenome DRAW tool (Lohse, Drechsel & Bock, 2007 (link)).