Sequel sequencing kit 3

High molecular weight (HMW) DNA from D. kikkawai (adult females), D. takahashii (adult females), and D. bipectinata (adult males and females) flies was provided by Dr. Bernard Kim at Stanford University. An overview of the DNA extraction protocol has previously been described (Kim, Miller, et al. 2021 (link); Kim, Wang, et al. 2021 (link)).
The library preparation and PacBio sequencing were performed by the McDonnell Genome Institute (MGI) at Washington University in St. Louis. The SMRTbell Express Template Prep Kit 2.0, Sequel Binding Kit 3.0, and Sequel Sequencing Kit 3.0 were used to prepare the samples for single-molecule real-time (SMRT) sequencing using the PacBio Sequel system. Each species was sequenced using one 1M SMRT cell in the continuous long-read (CLR) sequencing mode with the 6.0.0.45111 chemistry and a movie length of 600 min. The sequencing data were processed by version 7.0.1.66975 of PacBio SMRT Link.
To assess the quality of the PacBio sequencing data, the quality control (QC) tool in SequelTools (Hufnagel et al. 2020 (link)) and SEQUELstats were used to analyze the subreads and scraps BAM files for each species.

The concentration of HMW genomic DNA was measured using a Qubit Fluorometer dsDNA Broad Range assay (Thermo Fisher Scientific, Waltham, MA, USA). The CLR and HiFi library preparations started with 8 and 15 μg HMW DNA, respectively, using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA) according to the manufacturer's instructions (Supplementary Methods). The CLR SMRT bell template-polymerase complex was sequenced on a PacBio Sequel instrument using the Sequel Sequencing Kit 3.0 (PacBio, Menlo Park, CA, USA) with 6 Sequel™ SMRT® Cells 1M v3 (PacBio, Menlo Park, CA, USA), taking a 10-hour movie per cell. The HiFi SMRT bell template-polymerase complex was sequenced on a PacBio Sequel II instrument (PacBio Sequel II System, RRID:SCR_017990) using the Sequel II Sequencing Kit 2.0 (PacBio, Menlo Park, CA, USA) and 1 Sequel™ II SMRT Cell 8M (PacBio, Menlo Park, CA), taking a 30-hour movie.

We extracted 30 μg of high-quality genomic DNA from white blood cells of the male cynomolgus macaque using blood and cell culture DNA kits (QIAGEN). Double-stranded DNA was fragmented, and the size distribution of the sheared DNA was characterized using the DNA 12,000 kit on the Agilent 2100 BioAnalyzer System. DNA fractions of approximately 15 kb were size selected for sequencing. PacBio-CCS sequencing libraries were prepared using the SMRTbell Template Prep Kit v.1.0 (Pacific. No. 100-259-100), according to the manufacturer’s protocol. Four SMRT flow cells were run on the PacBio Sequel II System with the Sequel Sequencing Kit 3.0 chemistry (Pacific Biosciences Ref. No.101-500-400 and 101-427-800) at BGI-Qingdao.

DNA was extracted from overnight cultures (OD_600nm = 1.5) using PowerSoil DNA Isolation Kit (Qiagen, Hilden, Germany). After quantification by gel electrophoresis and fluorimetric assay (Qubit, Thermo Fisher Scientific, Waltham, MA, USA), we followed the procedure already reported in [25 (link)] for fragmenting DNA with g-TUBE (Covaris Inc., Woburn, MA, USA) to an average 15 kbp size and preparing the sequence library using the Pacific Biosciences SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA). Sequencing was performed on a Sequel apparatus (Pacific Biosciences, Menlo Park, CA, USA) by SMRT technology [21 (link)], using Sequel Sequencing Kit 3.0.

A region containing the variants of interest in IFT172 was directly amplified from genomic DNA using TaKaRa LA Taq DNA Polymerase with 10x LA PCR Buffer II (TaKaRa, Mountain View, CA) with a two-step PCR protocol (primers, S2 Table). Initial denaturation at 94°C for 1min was followed by 30 cycles of 98°C for 10s denaturation and 64.1°C for 22min and 30s elongation. Final elongation was performed by incubation at 72°C for 10min. Product length was 22,800 bp. LR-PCR products were purified using SPRI magnetic beads (Beckman Coulter Life Sciences, Brea, CA) according to the manufacturer’s protocol, with 2μl used for Qubit 2.0 Fluorometric Quantitation (Beckman Coulter Life Sciences) dsDNA high sensitivity assay. Purified samples were sequenced by Pacific Biosciences Sequel system (PacBio, Menlo Park, CA) according to manufacturer’s protocol using SMRTbell Express Template Kit 2.0 and Sequel Sequencing Kit 3.0. To obtain highly accurate reads, Circular Consensus Sequence (CCS) analysis was performed using Pacific Biosciences SMRT Link (v6.0). CCS reads were aligned to human reference genome (hg19) using Minimap2 v. 2.24 [15 (link)], sorted with SAMtools and visualized in IGV browser.

Genomic DNA was isolated from the kidney cortex of CDK12^RTEC+/- mice using a SMARTer PCR cDNA Synthesis Kit (Clontech, USA) according to the manufacturer's protocol. Library preparation and sequencing were conducted by OE Biotech Co., Ltd., (Shanghai, China) using SMRTbellTM Template Prep Kit 1.0-SPv3, Sequel Binding and Internal Ctrl Kit 3.0, Sequel Sequencing Kit 3.0 and SMRT Cell 1 M v3 LR Tray (Pacific BioSciences, Melon Park, CA, USA). High-quality RNAs were used to construct an Iso-Seq library. Briefly, a total of 12 PCR cycles of amplification were performed using PrimeSTAR GXL DNA Polymerase (Clontech, USA). After purification with AMPure® PB magnetic beads, the cDNA products were then subjected to the construction of SMRTbell template libraries using the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences, USA). Finally, the purified SMRTbellTM templates were bound and sequenced on the Pacific Bioscience Sequel System platform. Sequencing reads were subjected to circular consensus sequences (CCSs) using SMRT Analysis Software (https://www.pacb.com/products-and-services/analytical-software/devnet/). Integrative Genomics Viewer (IGV) was used to visualize PacBio long-read sequence data.