After slpH-specific PCR, amplicons were purified using the Qiaquick PCR Purification kit (Qiagen, Hombrechtikon, Switzerland). Purified amplicons were fragmented by sonication of 1 μg of DNA in 130 μL nuclease-free water at 50 W, 200 cycles/burst, and 20°C for 90 s on a Covaris M220 instrument (Covaris, Brighton, U.K.). The fragmented DNA was end-repaired and adapter-ligated using the Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific) according to the manufacturer's instruction. Samples were barcoded using the Ion Xpress Barcode Adapter 1-16 kit (Thermo Fisher Scientific). After barcoding, the DNA was size-selected for 400 bp reads using an E-Gel® SizeSelect™ Agarose Gel (Thermo Fisher Scientific). The libraries were quantified by qPCR using the Ion Library Quantitation Kit (Thermo Fisher Scientific). Before preparing the template-positive ion sphere particles with the Ion PGM Hi-Q OT2 kit (Thermo Fisher Scientific), each library was diluted to 100 pM in 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA, before being pooled. The sequencing was performed using either an Ion 314™ or 316™ chip and the Ion PGM Hi-Q Sequencing kit on an Ion Torrent sequencer (Thermo Fisher Scientific).
M220 instrument
Manufactured by Covaris
Sourced in United States
The M220 instrument from Covaris is a laboratory equipment designed for sample preparation. It utilizes advanced technologies to perform precise and controlled sample processing. The core function of the M220 is to facilitate efficient and reproducible sample preparation for a variety of applications.
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Lab products found in correlation
Kapa hyper prep kit, by Roche (7 mentions)
Agencourt ampure xp bead, by Beckman Coulter (5 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Hiseq 2000, by Illumina (2 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (2 mentions)
Biomek i7, by Beckman Coulter (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Miseq 2, by Illumina (2 mentions)
Hiseq 4000 platform, by Illumina (2 mentions)
Hiseq 4000 ngs platforms, by Illumina (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Ion pgm hi q sequencing kit, by Thermo Fisher Scientific (1 mentions)
Ion pgm hi q ot2 kit, by Thermo Fisher Scientific (1 mentions)
Ion xpress plus fragment library kit, by Thermo Fisher Scientific (1 mentions)
Ion xpress barcode adapter 1 16 kit, by Thermo Fisher Scientific (1 mentions)
Ion library quantitation kit, by Thermo Fisher Scientific (1 mentions)
Ion torrent sequencer, by Thermo Fisher Scientific (1 mentions)
26 protocols using m220 instrument
1
Ion Torrent Targeted Sequencing
2
Sugar Beet Germplasm Genotyping Protocol
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Seeds from a total of 606 Beta accessions were obtained from the public germplasm repositories of the USDA (https://www.ars-grin.gov/ ) and the IPK (https://www.ipk-gatersleben.de/ ), from three sugar beet breeding companies (KWS SAAT SE, Syngenta, Strube Research GmbH), and from the USDA sugar beet breeding program at Michigan State University, East Lansing, MI, USA. All plants were grown in the greenhouse at KWS, and leaf material was harvested from one single plant per accession approximately six weeks after sowing. Plant genomic DNA for genotyping and sequencing was extracted by a silica-membrane method using the NucleoSpin 96 Plant II kit (Macherey-Nagel, Düren, Germany). Genotyping data were produced with the Illumina Infinium HD-chip workflow according to the manufacturer´s instructions (Illumina, San Diego, CA, USA). Following quantification of genomic DNA by Qubit fluorometry (Life Technologies, Foster City, CA, USA), 200 ng of each DNA sample was sheared to a peak size of 580 bp with a Covaris M220 instrument (Covaris, Woburn, MA, USA).
3
Genomic DNA Extraction and Sequencing of Primate Lice
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Total gDNA was extracted from chimpanzee, gorilla, and human pubic lice using the Zymo Genomic DNA-Tissue MicroPrep kit following the manufacturer’s instructions except that lice were manually macerated prior to incubation with proteinase-K. Total gDNA from red colobus monkey lice was extracted using a phenol–chloroform method following Boyd et al. (2014) . DNA extracts from the Netherlands human head lice were provided by Ascunce (see Ascunce et al. 2013 (link) for collection methods).
gDNA from each louse was sonicated using the Covaris M220 instrument to an average fragment size of 300–450 bp (actual range was 200–600 bp). The sheared gDNA was prepared for next-generation sequencing using TruSeq DNAseq or Kapa Library preparation kits. The resulting library was sequenced on one-half lane of Illumina HiSeq2000 or 2500 using the TruSeq SBS sequencing kit v.1-2 for 161 cycles. All samples were sequenced paired-end, with 100 or 160 bp reads. Fastq files were produced using Casava v.1.8.2. Sequence data for the Uganda chimpanzee louse were obtained from the Genbank Short Read Archive (accession SRX390495; see Boyd et al. 2014 and Johnson et al. 2014 (link) for complete sequencing methods).
gDNA from each louse was sonicated using the Covaris M220 instrument to an average fragment size of 300–450 bp (actual range was 200–600 bp). The sheared gDNA was prepared for next-generation sequencing using TruSeq DNAseq or Kapa Library preparation kits. The resulting library was sequenced on one-half lane of Illumina HiSeq2000 or 2500 using the TruSeq SBS sequencing kit v.1-2 for 161 cycles. All samples were sequenced paired-end, with 100 or 160 bp reads. Fastq files were produced using Casava v.1.8.2. Sequence data for the Uganda chimpanzee louse were obtained from the Genbank Short Read Archive (accession SRX390495; see Boyd et al. 2014 and Johnson et al. 2014 (link) for complete sequencing methods).
4
Chromatin Immunoprecipitation of H3K9ac
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For each sample, chromatin was prepared from 10 mg hippocampal tissue using the TruChIP tissue kit (Covaris, Woburn, MA) and sheared using a Covaris M220 instrument according to optimized conditions as described previously (Ribeiro et al., 2021 (link)). From each sample, 4% of total chromatin was retained, and crosslinks were reversed to provide an input control. The remaining chromatin was divided into two equal parts, and chromatin immunoprecipitation was carried out in ChRIPA buffer after sequestering SDS, employing either an anti–histone 3 lysine 9 (H3K9ac) antibody (39137, Active Motif, Carlsbad, CA) or a rabbit IgG isotype control (ab171870, Abcam) in accordance with previously established protocols (Ribeiro et al., 2021 (link)). The immunoprecipitated DNA was subsequently assessed for H3K9ac occupancy at the Bdnf promoter IV region using quantitative polymerase chain reaction. The primers and conditions for the quantitative polymerase chain reaction were as previously described (Seo et al., 2016 (link); Asp, 2018 (link); Ribeiro et al., 2021 (link)).
5
Metagenomic DNA Extraction and Sequencing
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To generate metagenomic datasets for both FBT samples, genomic DNA was extracted from 0.5 g of prepared samples using the Fast DNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, United States) according to the manufacturer’s protocol. The concentration and purity of DNA were quantified with a TBS-380 mini-fluorometer and NanoDrop 2000 spectrometer, respectively (Li et al., 2020 (link)). DNA was then sheared into approximately 400-bp fragments using a Covaris M220 instrument. The metagenomic libraries were subsequently prepared using the NEXTFLEXTM Rapid DNA-Seq Kit (Illumina, San Diego, CA, United States). Paired-end sequencing was performed on the Illumina HiSeq 4000 platform at Majorbio Bio-Pharm Technology Co., Ltd., using a NovaSeq and HiSeq X Reagent Kits according to the manufacturer’s instructions.
6
Whole-genome Library Preparation and Sequencing
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Extracted genomic DNA samples were fragmented using a Covaris M220 instrument (Covaris, Inc., Woburn, MA) to ∼450 bp. Whole-genome libraries were prepared using a TruSeq Nano DNA kit with the Illumina NeoPrep system (Illumina, San Diego, CA), by following the kit instructions. Samples were sequenced using an Illumina MiSeq in 2 × 300 bp mode with 600-cycle V3 sequencing kits to a depth of ∼40× to 120× (see Table S1 in the supplemental material).
7
Genomic Profiling of Tumor Samples
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Fresh frozen tissues from tumor samples were used for genomic DNA extraction with QIAamp DNA FFPE Tissue Kit (QIAGEN) following the manufacturer’s instructions. White blood cell DNA was sequenced together with tumor DNA samples to identify germline mutations. The DNA quality was assessed by NanoDrop 2000 (Thermo Fisher Scientific) and the quantity was measured by dsDNA HS Assay Kit (Life Technologies) on Qubit V.2.0. Extracted tumor genomic DNA was fragmented into 300~350 bp using the Covaris M220 instrument (Covaris). Sequencing libraries were prepared with KAPA HyperPrep kit (KAPA Biosystems) with optimized protocols. DNA libraries from different samples were marked with unique indices during library preparation and up to 2 µg of different libraries were pooled together for targeted enrichment. The enriched libraries were sequenced on HiSeq 4000 NGS platforms (Illumina) to coverage depths of 200×. Trimmomatic was used for FASTQ file quality control. GATK V.3.4.0 was applied to detect germline mutations from blood control samples. VarScan V.2 was employed for detection of somatic mutations. Annotation was performed using ANNOVAR using the hg19 reference genome. TMB was defined as the total single nucleotide variant (SNV) counts in coding regions14 (link) and neoantigen was analyzed by NeoPredPipe (Neoantigen Prediction Pipeline).15 (link)
8
Chloroplast Genome Sequencing of Medicinal Plants
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The fresh leaves of Mitrephora tomentosa and Desmos chinensis were collected from the South China Botanical Garden, Chinese Academy of Sciences (coordinates: E113°36.9’, N23°18.1’, and E113°36.4’, N23°18.5’), and stored at -80°C. The total DNA was extracted using the E.Z.N. A.® Plant DNA kit (OMEGA, USA), and the DNA was fragmented using a Covaris M220 instrument. After purification, TruSeq™ RNA sample Prep Kit was used to construct the sequencing library. Paired-end sequencing was performed on the Illumina NovaSeq6000 platform with a read length of 150 bp. The raw data were filtered using Cutadapt v1.16 software (Martin, 2011 (link)), followed by assembly using GetOrganelle V1.7.7.0 software (Jin et al., 2020 (link)) with the seed set as gi|12394|emb|V00171.1| Zea mays chloroplast gene for the large subunit of RUBP (ribulose bisphosphate carboxylase), and the database utilized was embplant_pt. The genome was annotated using PGA (Qu et al., 2019 (link)) and Geseq (https://chlorobox.mpimp-golm.mpg.de/geseq.html ) web-based programs (Tillich et al., 2017 (link)), and the sequence was deposited in the NCBI database (https://www.ncbi.nlm.nih.gov ), accession numbers: OQ682535 and OQ687053. The plastome map was drawn online using OGDRAW software (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html ) (Greiner et al., 2019 (link)).
9
Sequencing Library Preparation Protocol
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Sequencing libraries were prepared using the KAPA Hyper Prep kit (KAPA Biosystems) with an optimized manufacturer’s protocol. In brief, 1 μg of genomic DNA, which was sheared into 350 bp fragments using the Covaris M220 instrument (Covaris), or 2–100 ng of cfDNA, underwent end-repairing, A-tailing and ligation with indexed adapters sequentially, followed by size selection using Agencourt AMPure XP beads. Finally, libraries were amplified by PCR and purified for target enrichment.
10
Targeted Gene Enrichment and Deep Sequencing
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Library preparation for each sample was performed according to the manufacturer’s protocol. Briefly, ~ 1 μg DNA was randomly sheared into 150–200-base pair fragments using a Covaris M220 instrument (Woburn, MA, USA), followed by library construction with a KAPA Hyper DNA Library Prep Kit (KAPA Biosystems, Wilmington, MA, USA). The adaptor library was amplified and linked, and the total library was accurately quantified by Qubit DNA HS Assay Kit (Invitrogen, CA, USA). A library hybridization kit, SeqCap EZ MedExome Enrichment kits (Roche, Basel, CH), was used to capture target sequences and bead capture and elution hybridization libraries with Roche’s customized 1000 targeted gene probes (Roche, Basel, CH). To construct the targeted gene list, we referred to FoundationOne and Integrated Mutation Profiling of Actionable Cancer Targets (IMPACT), which were designed by two authoritative organizations, Foundation Medicine and Memorial Sloan Kettering Cancer Center (MSK), respectively, and all received FDA approval.
After amplifying the captured library by PCR, the constructed library was sequenced by an Illumina HiSeq Xten sequencer (San Diego, CA, USA). The average sequencing depth of tissue samples was 500 X. It could detect mutations with very low frequency to 0.1%.
After amplifying the captured library by PCR, the constructed library was sequenced by an Illumina HiSeq Xten sequencer (San Diego, CA, USA). The average sequencing depth of tissue samples was 500 X. It could detect mutations with very low frequency to 0.1%.
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