Sequel 2 platform

Plants of P. ludlowii were grown in the national Tibetan Plateau crop germplasm garden at an altitude of ~3600 m. Young leaves from one plant were collected for DNA extraction and sequencing library preparation. One short-read paired-end library was constructed and sequenced with a read length of 150 bp on an Illumina Hiseq system. For PacBio HiFi sequencing, a high-molecular-weight DNA library was prepared using SMRTbell Express Template Prep Kit 2.0 and sequenced on a PacBio Sequel II platform. For RNA-seq, libraries were constructed from six different tissues, including roots, fruits, petals, buds, leaves, and branches, and sequenced on a NovaSeq 6000 platform. The fresh-year young roots and branches were sampled. The buds and young leaves were sampled at the flowering stage. The petals were sampled at the early flowering stage. The fruits were sampled 25, 50, 75, 100, 125, and 150 days after the end of the flowering stage, respectively, and mixed for RNA library construction. For Hi-C sequencing, young leaves of one plant were collected to extract high-quality genomic DNA samples. The samples were digested with 200 U DPN II restriction enzyme (Qiagen) for library construction. Hi-C libraries were controlled for quality and sequenced on an Illumina Novaseq platform with the model of 150 bp paired-end reads.

Multiple wild C. nippona individuals were collected from Zhoushan, Donghai Sea, China. The oysters were identified on the basis of both DNA fragments of cytochrome oxidase I (COI) and morphological observation. The samples were dissected, immediately frozen in liquid nitrogen, and stored at − 80 ℃ for further analysis. Genomic DNA was extracted from the adductor muscle of a male individual (674.3 g) by the standard phenol–chloroform method. A PacBio library (15–20 kb) was prepared using the SMRTbell Express Template Prep Kit 2.0. Single-molecule real-time sequencing was carried out on a PacBio Sequel II platform under the CCS mode. Then, the subreads were filtered by minimum length of 50 kb, and the HiFi reads were generated using ccs software (v 4.2.0) (https://github.com/PacificBiosciences/ccs) with the parameters of “min-passes = 3, min-rq = 0.99”. For genome survey, Illumina library was constructed using the DNA from the same oyster, and sequenced by a PE150 strategy on Illumina NovaSeq 6000 system. For Hi-C sequencing, the adductor muscle from the same individual for DNA extraction was fixed with 1% formaldehyde, and DNA was cross-linked and digested with MboI restriction enzyme. The library was also sequenced on an Illumina NovaSeq 6000 platform with a PE150 method.

We used SMRT sequencing to precise determine the rearrangement of β-globin gene cluster. Experiments were conducted by Berry Genomic Corporation (Beijing, China) as described in previous study^{9 (link)}. Briefly, genomic DNA was subjected to PCR with primers covering the majority of known structural variations, SNVs and indels in HBA1, HBA2 and HBB regions, as well as the DNA region encompassing the duplication probes by MLPA. PCR products were ligated with barcoded adaptors by a one-step end-repair and ligation reaction to construct pre-libraries, which were pooled together by equal mass and converted to single-molecule real-time dumbbell (SMRTbell) library by Sequel Binding and Internal Ctrl Kit 3.0 (Pacific Biosciences). Then, SMRTbell library was sequenced under circular consensus sequencing (CCS) mode on Sequel II platform (Pacific Biosciences). The converted CCS reads were aligned to hg38 and the precise regions of duplication were determined.

The de novo genome sequencing was performed on Sequel II platform of Pacific Biosciences (PacBio), and the next generation sequencing was performed on an Illumina NovaSeq PE150 platform. The genome was assembled using SMRT Link v5.0.1 software and corrected with next generation sequencing data [65 (link)]. Finally, the accuracy and integrity of the assembled genome were examined using the Core Eukaryotic Genes Mapping Approach (CEGMA) and Benchmarking Universal Single-Copy Orthologs (BUSCO) [66 (link), 67 (link)].

Samples were sent to Berry Genomics for CATSA sequencing. Previous multiplex long PCR analysis (Liang et al., 2021 (link)) was modified to amplify additional genomic regions including HBD and HS40 regions. The region analyzed for HS40 was chr16: 109217–117302. The region analyzed for HBA1 and HBA2 was chr16: 169296–178914. The region analyzed for HBB and HBD was chr11: 5223675–5235660. Then, a barcoded adaptor was ligated to the PCR products by a one-step end-repair and ligation reaction. Unligated products were removed by exonucleases (Enzymatics), and pre-libraries were pooled together by equal mass. Next, the SMRT bell library was generated from the pooled library using Sequel Binding and Internal Ctrl Kit 3.0 (Pacific Biosciences) and sequenced using the CCS mode on the Sequel II platform (Pacific Biosciences). The raw subreads were converted to CCS reads by CCS software (Pacific Biosciences) and demultiplexed by barcodes using lima in the Pbbioconda package (Pacific Biosciences). The demultiplexed CCS reads were then aligned to genome build hg38 with pbmn2. FreeBayes1.3.4 (https://www.geneious.com/plugins/freebayes; Biomatters, Inc., San Diego, CA) was used to call SNVs and indels. Structural variations were called based on HbVar, Ithanet, and LOVD databases.

Experiments were performed as previously described^{14 (link)}. Briefly, genomic DNA was amplified by PCR with primers targeting the majority of known structural variations, SNVs and indels in the HBA1, HBA2 and HBB genes. Barcoded adaptors were ligated to the PCR products to construct individual sequencing libraries. Then, each library was quantified and pooled together by equal mass. After purification and quantification, the pooled library was converted to a SMRTbell library with Sequel Binding and Internal Ctrl Kit 3.0 (Pacific Biosciences) and sequenced on the Sequel II platform (Pacific Biosciences) under CCS mode. Then raw subreads were analyzed by CCS software (Pacific Biosciences) to generate CCS reads, debarcoded by lima in the Pbbioconda package (Pacific Biosciences) and aligned to genome build hg38 by pbmn2 (Pacific Biosciences). Finally, structural variations were identified according to the HbVar, Ithanet and LOVD databases. SNVs and indels were identified by FreeBayes1.3.4 (https://www.geneious.com/plugins/freebayes; Biomatters, Inc., San Diego, CA).