RAD sequencing library preparation and sequencing was conducted by the Novogene Bioinformatics Technology CO. ltd, Beijing. Briefly, the libraries were prepared following DNA digestion with EcoRI, random fragmentation with the Covaris S220 instrument (Covaris, Woburn, MA, USA), barcode ligation and DNA purification, gel fragment selection, adapter ligation and fragment amplification. Pair-end sequencing with a read length of 150 bp was used to produce approximately 4 Gb of raw data for each sample with the Illumina HiSeq. 2000 platform (Illumina, San Diego, CA, USA).
S220 instrument
Manufactured by Covaris
Sourced in United States
The Covaris S220 instrument is a high-performance sample preparation device designed for efficient and reproducible processing of biological samples. It utilizes advanced acoustic technology to uniformly disrupt samples, enabling effective extraction, homogenization, and fragmentation of materials. The S220 instrument provides a controlled and consistent sample processing experience, ensuring reliable results for a variety of applications.
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Lab products found in correlation
Hiseq 1000, by Illumina (3 mentions)
Hiseq 2000, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Sureselect human all exon v4 utrs lincrna, by Agilent Technologies (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Nextflex dna sequencing kit, by PSC Biotech (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Miseq platform, by Illumina (2 mentions)
Ez dna methylation lightning kit, by Zymo Research (2 mentions)
Bioo nextflex bisulfite seq kit, by Illumina (2 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
V3 600 cycle kit, by Illumina (1 mentions)
Agilent rna 6000 pico kit, by Agilent Technologies (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Hiseq 2000 instrument, by Illumina (1 mentions)
Agilent qpcr ngs library quantification kit, by Agilent Technologies (1 mentions)
Sureselect xt target enrichment system for paired end sequencing library protocol, by Illumina (1 mentions)
29 protocols using s220 instrument
1
RAD Sequencing Library Preparation
2
Fragmentation and Library Preparation for Metatranscriptome Sequencing
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The enriched mRNA was mechanically fragmented to a size range of ~200 bp with an ultrasonicator using the adaptive focused acoustics following the manufacturer recommended protocols (Covaris S220 instrument, Covaris Inc.). The fragmentation of mRNA was assessed using Agilent RNA 6000 Pico Kit on 2100 Bioanalyzer instrument (Agilent Technologies, Inc.). The metatranscriptome libraries were prepared using NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs Inc). The quality and quantity of all the final libraries were analyzed with an Agilent DNA 1000 Kit on the 2100 Bioanalyzer Instrument and Qubit. The final libraries were quantitated and validated by qPCR assay using the PerfeCTa NGS Library Quantification Kit for Illumina (Quanta Biosciences, Inc.) using the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). Sequencing of one of the MT library was performed on a Illumina HiSeq 2000 using the TruSeq SBS v3 reagent for paired-end 100 read length (BGI Americas) (labeled as HS100), and on Illumina MiSeq using v3-600 cycle kit for paired-end 301 bases (labeled as MS301). Another set of twelve libraries was sequenced on Illumina MiSeq using 151 paired end chemistry (labeled as MS151). Manufacturer's recommended protocol was used for performing the sequencing reaction on both the HiSeq and MiSeq platforms.
3
High-throughput DNA sequencing library preparation
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Genomic DNA was fragmented into segments of 150–300 bp by a Covaris S220 instrument (Covaris, Inc., Woburn, MA, USA). The DNA libraries were created using a KAPA Hyper Prep Kit (Kapa Biosystems, Boston, MA, USA), followed by Agilent’s SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library Protocol (Agilent Technologies, Santa Clara, CA, USA). The DNA libraries were quantified by an Agilent QPCR NGS Library Quantification Kit (Agilent Technologies), and DNA libraries with average insert sizes of 150 bp were sequenced on an Illumina HiSeq 2000 instrument (Illumina, San Diego, CA, USA).
4
High-Throughput Whole Genome Sequencing
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NEBNext® Ultra™ II DNA Library Prep Kit for Illumina clustering and sequencing reagents were used according to the manufacturer's recommendations. Briefly, the genomic DNA was fragmented by acoustic shearing with a Covaris S220 instrument. Fragmented DNA was cleaned and end-repaired (Supplementary Figure 2B ). Adapters were ligated after adenylation of the 3′ ends followed by enrichment by limited-cycle PCR. DNA libraries were validated using a DNA 1000 Chip on the Agilent 2100 Bioanalyzer (Supplementary Figure 2C , Agilent Technologies, Palo Alto, CA, USA) and quantified using Qubit 2.0 Fluorometer. The DNA libraries were also quantified by real-time PCR (Applied Biosystems, Carlsbad, CA, USA), and multiplexed in equal molar mass. The pooled DNA libraries clustered on 10 lanes. After clustering, the samples were loaded on the Illumina HiSeq instrument according to the manufacturer's instructions. The samples were sequenced using a 2x 150 paired-end (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS) on the HiSeq instrument (Supplementary Figure 2D ). WGS was performed with Q30 bases coverage and over 94–95% of Aligned reads (Supplementary Figure 2D , Supplementary Table 2 ). The genomic data of each of the 10 cases were normalized to a reference genome from Homo sapiens (NCBI GRCh38 with decoys, female).
5
Sheared 3C Library Sequencing
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Approximately 5 µg of a 3C library was suspended in water (final volume 130 µl) and sheared using a Covaris S220 instrument (Duty cycle 5, Intensity 5, cycles/burst 200, time 60 s for 4 cycles). The DNA was purified using a Qiaquick® PCR purification kit, DNA ends were prepared for adapter ligation following standard protocols78 (link). Custom-made adapters23 (link) were ligated overnight at 4 °C. Ligase was inactivated by incubating the tubes at 65 °C for 20 min. To purify DNA fragments ranging in size from 400 to 900 pb, a PippinPrep apparatus (SAGE Science) was used. For each library, one PCR reaction of 12 cycles was performed using 2–3 µl of 3C library, 0.2 µM Illumina primers PE1.0 and PE2.0, and 1 unit of Taq Phusion (Finnzymes). The PCR product was purified on Qiagen MinElute columns and dimers of primers were removed from the 3C library by using AMPure XP beads following the manufacturer’s protocol (Beckman Coulter). Finally, libraries were subjected to 75 bp paired-end sequencing in an Illumina sequencer (NextSeq500).
6
RNA-Seq Library Preparation Protocol
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Total RNA (100 ng) was converted to cDNA using the NuGEN Ovation RNA-Seq System v2 (Cat. # 7102–32) (NuGEN) following the manufacturer’s protocol (NuGEN, San Carlos, CA). NuGEN-amplified double-stranded cDNAs were broken into ~ 180 base pair (bp) fragments by sonication with a Covaris S220 instrument (Covaris, Woburn, MA). Fragmented cDNAs were processed on a SPRI-TE library construction system (Beckman Coulter, Fullerton, CA). Uniquely indexed NEXTflex adapters (Bioo Scientific, Austin, TX) were ligated onto each sample to allow for multiplexing. Adapter-ligated libraries were amplified [1 cycle at 98 °C for 45 s; 15 cycles at 98 °C for 15 s, 65 °C for 30 s, and 72 °C for 30 s; 1 cycle at 72 °C for 1 min; and a hold at 4 °C] using a KAPA library amplification kit (KAPA Biosystems, Wilmington, MA) and purified with AMPure XP beads (Beckman Coulter).
7
DNA Extraction and Illumina Sequencing
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DNA was isolated as previously described [13 (link)] (see also extended methods in the Supplementary Material). The Qubit dsDNA High Sensitivity Kit (Thermo Fisher Scientific, Karlsruhe, Germany; #Q32854) was applied to measure DNA concentration. 10–50 ng of DNA was sheared into 300 bp fragments using a Covaris S220 instrument [13 (link)]. Fragment libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Frankfurt am Main, Germany; #E7645L) and NEBNext Multiplex Oligos for Illumina (#E6440) in six to eight PCR cycles. Purification of fragment libraries was performed with 0.9× AMPure Beads (Beckman Coulter, Krefeld, Germany; #A63881). The Illumina NextSeq 550 platform was applied for single-ended 75 bp short-read sequencing (Illumina, Cambridge, UK; #20024906). Negative controls were processed and sequenced in parallel with the patient samples.
8
Whole Genome Library and Exome Capture for Schistosoma mansoni
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We prepared whole genome libraries of F0s using the KAPA HyperPlus kit (KAPA Biosystem) according to the manufacturer’s protocol. For each F0 library, we sheared 500 ng of gDNA by adaptive focused acoustics (Duty factor: 10%; Peak Incident Power: 175; Cycles per Burst: 200; Duration: 180 seconds) in AFA tubes, using a Covaris S220 instrument with SonoLab software version 7 (Covaris, Inc., USA), to recover fragmented DNA between 150–200 bp. We used 6 PCR cycles for post-ligation library amplification.
We captured F1 and F2 (188 per cross) S. mansoni exomes using the SureSelectXT2 Target Enrichment System (Agilent). The design of the custom baits used to capture the S. mansoni exome (SureSelect design ID: S0398493) is described in Chevalier et al. [45 (link)], and exome capture methodology follows Le Clec'h et al. [52 (link)].
We sequenced the libraries on a HiSeq 2500 sequencer (Illumina) using 100 bp pair-end reads. On each sequencing lane, we either pooled 32 exome capture libraries or two whole genome libraries. Raw sequence data are available at the NCBI Sequence Read Archive under accession numbers PRJNA667697.
We captured F1 and F2 (188 per cross) S. mansoni exomes using the SureSelectXT2 Target Enrichment System (Agilent). The design of the custom baits used to capture the S. mansoni exome (SureSelect design ID: S0398493) is described in Chevalier et al. [45 (link)], and exome capture methodology follows Le Clec'h et al. [52 (link)].
We sequenced the libraries on a HiSeq 2500 sequencer (Illumina) using 100 bp pair-end reads. On each sequencing lane, we either pooled 32 exome capture libraries or two whole genome libraries. Raw sequence data are available at the NCBI Sequence Read Archive under accession numbers PRJNA667697.
9
Exome Capture and Sequencing Protocol
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Approximately 2.0 μg of genomic DNA from each cell sample was sonicated to give a fragment size of 200 bp on a Covaris S220 instrument. After 5–6 cycles of PCR amplification, capture and library preparation were performed with Agilent SureSelect Human All Exon V4 + UTRs + lincRNA (80 Mb), followed by washing, elution, and additional 10-cycle PCR. Enriched libraries were sequenced on an Illumina HiSeq 1000 operated in 101-bp paired-end mode. Image analyses and base calling on all lanes of data were performed using CASAVA 1.8.2 with default parameters.
10
Exome Sequencing of CTEPH Patients
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Library preparation and exome capture were performed using the SureSelectXT Target Enrichment System (Agilent Technologies, Santa Clara, CA) on a Bravo Automated Liquid Handling Platform (Agilent Technologies) as follows. Briefly, 1 μg of genomic DNA was sheared to 150‐ to 200‐bp fragments using a Covaris S220 instrument (Covaris, Woburn, MA), followed by end repair, A‐tailing, and adapter ligation. Precapture libraries were amplified by 6 cycles of polymerase chain reaction and analyzed using the Agilent 2200 TapeStation (Agilent Technologies) to evaluate quality and yield. Exome capture was performed with the SureSelectXT Human All Exon V5 Plus Regulatory kit (Agilent Technologies), followed by library amplification with 11 or 12 cycles of polymerase chain reaction. We assessed the quality of sequencing libraries by quantitative MiSeq methods.26 Paired‐end sequencing with 2×101 bp reads was performed on Illumina HiSeq 2500 (Illumina Inc, San Diego, CA). The mean output was 6.4 Gb per sample, and mean coverage depth was 38.8‐fold. We used the data from the integrative Japanese Genome Variation Database 3.5KJPN (https://ijgvd.megabank.tohoku.ac.jp/ ) to determine the allele frequency in the general population. Samples from 51 patients with CTEPH and the general population as references were obtained in the same area, eastern Japan, specifically in the Tohoku district.
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