For full genome sequences, 500 ng of input material were fragmented using Covaris M220 Focused-ultrasonicator™ (Covaris), and ligated to suitable Illumina adapters (NEXTflex-96™ DNA Barcodes, BiooScientific) using a SPRI-TE library system (Beckman Coulter) with SPRIworks Fragment Library Cartridges II (for Roche FLX DNA sequencer; Beckman Coulter). Size exclusion was performed manually with AMPure XP magnetic beads in two steps for a final size distribution of 500–600 bp long fragments. After quality control of the libraries on a Bioanalyzer 2100 (Agilent Technologies), the libraries were quantified using using Kapa Library Quantification Kit for Illumina platforms (Kapa Biosystems), pooled and sequenced on a MiSeq instrument (Illumina) with MiSeq reagent Kit v3 in 2 × 300 bp PE mode (Illumina). For data analysis, the reads were mapped against the nearest reference genome (Newbler v3.0, Roche). All mapped reads were extracted and de novo assembled (Newbler v3.0, Roche). Since this approach delivered three or more contigs, the software ContigGraph (unpublished) was used to determine the connections of single contigs for manual assembly of the full genome. Afterwards the whole data set was mapped against the full genome (Newbler v3.0, Roche).
M220 focused ultrasonicator
Manufactured by Covaris
Sourced in United States, United Kingdom
The M220 Focused-ultrasonicator is a laboratory instrument designed for sample preparation using focused acoustic energy. It is capable of disrupting the cellular structure of samples, enabling efficient extraction and analysis of biomolecules such as DNA, RNA, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (26 mentions)
2100 bioanalyzer, by Agilent Technologies (21 mentions)
Kapa hyper prep kit, by Roche (16 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (15 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (14 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (13 mentions)
Nextseq 500, by Illumina (13 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (13 mentions)
Kapa library quantification kit, by Roche (13 mentions)
Agencourt ampure xp bead, by Beckman Coulter (12 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (10 mentions)
Ampure xp bead, by Beckman Coulter (10 mentions)
Bioanalyzer, by Agilent Technologies (10 mentions)
Hiseq 4000, by Illumina (9 mentions)
Hiseq 2500, by Illumina (9 mentions)
Nextseq 500 sequencer, by Illumina (8 mentions)
Ez dna methylation gold kit, by Zymo Research (8 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (7 mentions)
Miseq platform, by Illumina (7 mentions)
Miseq benchtop sequencer, by Illumina (7 mentions)
322 protocols using m220 focused ultrasonicator
1
Whole Genome Assembly from Illumina Sequencing
2
Illumina Library Preparation from Viral DNA
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Accel-NGS 1S Plus DNA Library Kit for Illumina (Swift Biosciences, Ann Arbor, MI, USA) was employed to prepare sequencing libraries from isolated DNA of the viral enriched samples. This includes a shearing step at the beginning to yield the 2–300 bp sized DNA fragments using the Covaris M220 Focused-ultrasonicator (Covaris INC., Woburn, MA, USA). All subsequent reactions were carried out according to the manufacturer’s recommendations. Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to verify library DNA quality. Sequencing was performed on the Illumina HiSeq platform by GATC Biotech AG (Konstanz, Germany).
3
Mitochondrial Genome Sequencing and Annotation
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Genomic DNA was fragmented to 400–500 bp using a Covaris® M220 focused ultrasonicator (Covaris, Woburn, MA, USA). A 460 bp paired-end library was generated from each sample separately and finally sequenced using an Illumina HiSeq X Ten platform (Majorbio Bio-pharm Technology, Shanghai, China) to obtain 4 Gb of data. Reads with low sequencing quality were filtered out using Trimmomatic software (v0.36, http://www.usadellab.org/cms/?page=trimmomatic ). Sequences were removed if they: contained splice fragments, had low mass values (Q < 25), or had lengths of low mass bases greater than half the total sequence length. The software Spades (Center for Algorithmic Biotechnology, St. Petersburg, Russia) [25 ] was used to assemble clean reads, and the assembly results were compared by mapping to obtain the longest segment. The MitoZ program (BGI-Shenzhen, Shenzhen, China) [26 (link)] was used to annotate the assembled genome, and the coding sequences (CDS), transfer RNA (tRNA), and ribosomal RNA (rRNA) were identified. The mitochondrial genome sequences of all eight species were deposited at GenBank (see Table 1 for deposit numbers).
4
Cancer-related Gene Sequencing Protocol
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Genomic DNA were sheared into 150–200 bp fragments via Covaris M220 focused-ultrasonicator (Covaris, USA), and then KAPA HTP Library Preparation Kit (Illumina Platform) (KAPA Biosystems, USA) was utilized to establish fragmented DNA library. DNA library was captured by designed NimbleGen SeqCap EZ Library (Roche, USA) containing typical cancer-related genes, and then subjected to Novaseq 6000 for paired-end sequencing. Sequencing depth, DNA concentration and library input quantity were 471–9770, 0.02–79.4 ng/µl and 1–401 ng.
5
Genomic DNA Fragmentation and Library Preparation
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Genomic DNA was fragmented after quantification and quality assessment for library preparation. A total of 100 ng of DNA were diluted in 50 µL of 10 mM TRIS–HCl pH 8.5—Ethanol 20% (v/v) solution (Sigma–Aldrich Merck, Gillingham, UK) and sheared with Covaris M220 Focused-ultrasonicator (Covaris Ltd., Brighton, UK) with the following settings for a 350 bp insert size: duty factor (%) 20.0, peak power 50.0, cycles/Burst 200 and duration of 65 s at 20 °C. Libraries were prepared with a Truseq Nano DNA Library prep kit (Illumina, Cambridge, UK), according to the manufacturers’ instruction. Steps included fragmentation, repair ends and library size selection, addition of adenylate 3′ ends, ligation of adapters, enrichment of DNA Fragments, normalization and pool. The paired end libraries were generated with the Illlumina MiSeq at CABI (Egham, UK), with an average insert size of 350 bp and run on an Illumina MiSeq 250-PE V2 Cartridge (500 cycles) (Illumina, Cambridge, UK).
6
Whole Exome Sequencing Variant Filtering
7
TMB Calculation from NGS Panels
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Tumor mutational burden value was calculated from the high‐throughput sequencing data from both 295‐gene panel and 1021‐gene panel.14 , 15 The gene list for each panel is listed in Table S2 and S3 . The NGS library preparation and sequencing protocol were performed as follows. In brief, the genomic DNA was fragmented by Covaris M220 focused ultrasonicator (Covaris, Inc.), converted to an NGS library by end repair, A‐tailing, and adapter ligation. Then, DNA library was purified and quantified using Qubit 2.0 fluorimeter with the dsDNA high‐sensitivity assay kit (Life Technologies). The samples tested with 295‐gene panel were indexed and sequenced on Nextseq500 (Illumina, Inc.) and 1021‐gene panel indexed samples were sequenced on Gene+Seq‐2000 (Geneplus‐Beijing Institute) with paired‐end reads. In both 295‐gene and 1021‐gene panels, TMB was calculated by the number of somatic missense mutations, nonsense mutations, and coding indels and displayed as the number of mutations per Mb of captured genome. Fusions, copy number variations, and non‐coding mutations were not counted. According to x‐tile software,16 the optimal cutoff value used to define high TMB is 6.1 mutations/Mb in 295‐gene panel and 15.4 mutations/Mb in 1021‐gene panel, respectively.
8
Whole Exome Sequencing Variant Filtering
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Patient 1 was detected in a family-based WES study (unpublished data (CH, GV, Filip Van Nieuwerburg, NVdA, RFK)). Patient DNA was fragmented using Covaris® M220 Focused-ultrasonicator™ (Covaris, MA, USA), followed by TruSeq DNA Sample Preparation (Illumina Inc, San Diego, CA, USA), enrichment using the SeqCap EZ Human Exome Library v3.0 kit (NimbleGen, Roche, Penzberg, Germany), and sequencing on HiSeq 2000 (Illumina Inc, San Diego, CA, USA), all following standard protocols. Data analysis was performed using Galaxy (see URLs)49 (link)-51 (link). Variants were filtered by VariantDB (see URLs, manuscript in preparation) to exclude variants with (1) low quality, using thresholds based on correlation between NGS data and SNP-chip genotyping; (2) intronic or intergenic location, except splice sites; and (3) inheritance from the parents. WES sequencing of patients 2, 3 and 4 was performed as described2 (link),8 (link). The mutation in patient 5 was identified in a family trio based study. WES was performed using Illumina technology (Illumina Inc, San Diego, CA, USA), and sequence data was returned and analysed using software supplied from Oxford Gene Technology. Presence of reported (de novo) mutations were confirmed by an independent technique such as Sanger sequencing. Raw sequence data will be uploaded in The European Genome-phenome Archive (EMBL-EBI) database.
9
DNA Extraction and Shearing for NGS
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DNA from blood samples was extracted using the Maxwell® RSC Blood DNA Kit on the Maxwell® 16 Forensic Instrument (Promega, Mannheim, Germany) and eluted in a final volume of 50 µL. Per sample, four spots of 40 µL blood were extracted separately and the eluates were subsequently pooled to a total volume of 200 µL. DNA from saliva samples was extracted using Maxwell® FSC DNA IQ™ Casework Kit (Promega, Mannheim, Germany). Samples were quantified using Promega’s PowerQuant® System using 5 µL of PowerQuant 2 × MM, 0.5 µL of 20 × Primer/Probe/IPC-Mix, 3.5 µL HPLC, and 2 µL of the sample. Prior to NGS library construction, DNA was sheared mechanically to allow analysis of natural strand breaks at positions weakened due to depurination in future analyses of these datasets [18 (link)]. Shearing to 250 bp was performed following the manufacturer’s guideline for the Covaris® M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA) with a duty cycle of 10%, intensity 4, and 200 cycles per burst for 80 s. After shearing, the DNA was quality checked by 2100 Bioanalyzer Instrument with High Sensitivity DNA Kit (Agilent, Waldbronn).
10
Cancer-Related Gene Sequencing Protocol
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DNA extracted from the tumor, effusion, and LCO samples was fragmented using the Covaris M220 focused ultrasonicator (Covaris, Inc., Woburn, MA, USA) or Bioruptor ultrasonicator (diagenode sa, Inc., Seraing, Belgium), followed by end repair, phosphorylation, dA addition, and adaptor ligation for library construction. The samples were subsequently subjected to capture-based targeted sequencing using a panel of 520, 425, 168, or 139 cancer-related genes (panels of 520 or 168 belongs to Burning Rock Biotech, Guangzhou, China, while panels of 425 or 139 belongs to Nanjing Geneseeq Technology Inc.). DNA was hybridized with capture probe baits, selected with magnetic beads, and amplified using PCR. A bioanalyzer high-sensitivity DNA assay was performed to assess the quality and size of the fragments. Indexed samples were sequenced on the Nextseq500 sequencer (Illumina, Inc., San Diego, CA, USA) or HiSeq 4000 sequencer (Illumina, Inc., San Diego, CA, USA) with paired-end reads.
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