Genomic DNA was isolated from bacteria using the Qiagen QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). DNA libraries were prepared using a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA), followed by a 2 × 300 paired-end sequencing runs with 100× coverage using Illumina MiSeq equipment. Raw data generated on the MiSeq was further analyzed using tools available in the CLC Genomics Workbench Software, version 8.5 (Qiagen). Using the ‘Trim Sequences Tool’, sequence reads were trimmed to include quality trimming and ambiguity trimming, and length trimming to discard reads below a length of 50 bases. Trimmed reads were assembled using the ‘De novo Assembly Tool’; the assembly algorithm works by using de Bruijn graphs to produce contiguous (contig) sequences (minimum contig length was set at 200 bases).
Miseq equipment
Manufactured by Illumina
Sourced in United States
The MiSeq is a benchtop DNA sequencing system designed for targeted sequencing applications. It utilizes Illumina's proprietary sequencing-by-synthesis technology to generate high-quality sequence data. The MiSeq is capable of producing up to 15 gigabases of data in a single run, making it suitable for a range of applications such as targeted resequencing, small genome sequencing, and amplicon sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Sureselectxt target enrichment system capture ngs strategy library, by Agilent Technologies (4 mentions)
Nextera xt dna library preparation kit, by Illumina (2 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Precellys 24 tissue lyser, by Bertin Technologies (1 mentions)
Quant it picogreen double stranded dna dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Ion torrent s5, by Thermo Fisher Scientific (1 mentions)
Ion ampliseq custom panel, by Thermo Fisher Scientific (1 mentions)
Ion ampliseq designer, by Thermo Fisher Scientific (1 mentions)
Miseq reagent kit v2 500 cycle, by Illumina (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Library quantification kit, by Roche (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Pcr purification kit, by Illumina (1 mentions)
Kapa high fidelity polymerase, by Roche (1 mentions)
Nextera xt kit, by Illumina (1 mentions)
Access array system, by Standard BioTools (1 mentions)
Miseq sequencer, by Illumina (1 mentions)
Truseq dna pcr free library preparation kit, by Illumina (1 mentions)
25 protocols using miseq equipment
1
Bacterial Genomic DNA Sequencing Protocol
2
Microbiome Profiling via Targeted Amplicon Sequencing
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Root pool samples were homogenized by grinding in a mortar filled with liquid nitrogen and treatment with a Precellys24 tissue lyser (Bertin Technologies) for two cycles at 5,600 rpm for 30 s. DNA was extracted with the FastDNA spin kit for soil, according to the manufacturer’s protocol (MP Bioproducts). DNA concentrations were measured fluorometrically (Quant-iT PicoGreen double-stranded DNA [dsDNA] assay kit; Life Technologies, Darmstadt, Germany) and adjusted to 3.5 ng/μl. Barcoded primers targeting the variable V5-V7 region of the bacterial 16S rRNA gene (799F and 1193R [29 (link)]) or targeting the ITS2 region of the eukaryotic ribosome (fITS7 and ITS4 [30 (link), 31 ]) were used for amplification. The amplification products were purified, pooled, and subjected to sequencing with Illumina MiSeq equipment.
3
Genetic Analysis of Familial Dementia
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DNA was extracted from peripheral blood leukocytes using the QIAamp DNA blood Mini-kit (Qiagen). The genetic study of the proband (I.3) included a NGS panel with the coding region of the following genes: PSEN1, PSEN2, APP, MAPT, PGRN, VCP, CHMP2B, TARDBP, FUS, ADAM10, SORL1, SNCA, TREM2, UBIQLN2, ITM2B, CSF1R, TYPOBP, and SQSTM1 (amplified by Ampliseq technology and sequenced using a Miseq equipment, Illumina, yielding a coverage of over 200x). The APOE genotype and the hexanucleotide expansion in intron 1 of C9ORF72 were also analyzed. Potential pathogenic variants were reviewed in genomic databases (dbSNP, ExAc, Genome Aggregation Database, and Exome Variant Server) and analyzed for pathogenicity with prediction programs. Potentially pathogenic variants in the proband and siblings were confirmed by Sanger sequencing (Big Dye v3.1; ABI 3730, Applied Biosystems). The segregation pattern was analyzed in the family. After identifying the p.Tyr167* mutation, we further screened for this particular ADAM10 variant in another 197 familial DAT samples from the same cohort, 200 controls from the same population background, and 274 AD cases from an independent Spanish cohort, provided by the Banco Nacional de ADN Carlos III (Salamanca University).
4
Viral RNA Sequencing from Infected Cells
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Cells were infected in the presence or in the absence of NH4Cl (MOI of 0.01 PFU/cell) in triplicate wells. Viral RNA was extracted from each sample and cDNA was synthesized. A fragment of 463 nucleotides in length comprised between the C terminus of VP3 and the N terminus of VP1 was amplified using oligonucleotide primers CGGGACAATCAACCTACAC GTTGGTTATCCGACACTG coupled to nucleotide adapters CS1/CS2. Library preparation, amplification and sequencing were performed at the Parque Científico de Madrid using a MiSeq equipment (Illumina, San Diego, CA, USA) using the 2 × 300 bp MiSeq Reagent Kit v3. A total of 6231577 were analyzed (average reads per sample 519,298 ± 122,251).
5
Mutational Analysis of B-cell Lymphoma Genes
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The mutational status of 66 B-cell lymphoma–related genes (supplemental Table 1) was investigated using a SureSelectXT Target Enrichment System Capture NGS strategy library (Agilent Technologies) before sequencing in a MiSeq equipment (Illumina, San Diego, CA) (supplemental Methods). As verification, 25 cases were additionally investigated with a 10-gene Ion AmpliSeq Custom panel (supplemental Table 2) (Ion Torrent S5; Thermo Fisher Scientific). The Custom Panel was designed using the Ion AmpliSeq Designer from Thermo Fisher Scientific (version 3.4). For description of library preparation, sequencing, and raw data analyses see supplemental Methods. The previously published mutational profile of 17 pediatric and young adult cases of LBCL-IRF4 was used for comparison.20 (link) Occurrence of mutations in genes within predefined pathogenic pathways was calculated per each molecular group.26 (link)
6
Metagenomic Analysis of Gut Microbiome
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Stool samples were thawed, and the bacterial DNA were extracted and purified using the QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The 16S rRNa amplicons were generated by PCR with primers targeting the V2 region of the gene using a Pfx polymerase enzyme (Thermo Fisher Scientific, Waltham, MA, USA). The primers were designed with Illuminas©’ adapters. Amplicons were purified with AMPure beads and sequenced using a Miseq v2 500 cycle reagent kit, which generates paired end reads of 250 bp, in a Miseq equipment (Illumina, San Diego, CA, USA).
7
Amplicon Sequencing of Phage Inserts
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Sequencing of phage inserts was performed as described (Tang et al., 2019 (link)). In brief, plasmid DNA (10 ng) from the unselected library and from the third rounds of selection (control, asymptomatic, cardiomyopathy) was amplified by PCR with specific primers (Table S4 ). Four different forward and reverse primers were used (containing zero to three degenerated bases to add the diversity necessary for amplicon sequencing with the Illumina platform). The primers also contained an overhang corresponding to the sequence recognized by the Nextera XT kit. Phage were amplified for 20 cycles (melting: 95°C for 30 s; annealing: 55°C for 30 seconds; extension: 72°C for 1 min) with Kapa high-fidelity polymerase (Kapa Biosystems). All PCR products were purified (QIAGEN PCR purification kit) and a second PCR was performed to add the index adaptors (barcodes) by using the Nextera XT kit (Illumina) following a standard manufacturer protocol. The resulting libraries were quantified by qPCR by using the library quantification kit (Kapa Biosystems), diluted (4 nM), denatured (0.2M NaOH and 95°C for 5 min), and sequenced with the MiSeq Reagent Kit v2 (500 cycles) on an Illumina MiSeq equipment.
8
Comprehensive Mutational Profiling of B-cell Lymphomas
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The mutational status of all cases was studied with a panel of 68 B-cell lymphoma–related genes (Supplementary Table 2, Supplemental Digital Content 2, http://links.lww.com/PAS/B442 ) using a SureSelectXT Target Enrichment System Capture NGS strategy library (Agilent Technologies) before sequencing in a MiSeq equipment (Illumina). The average sequencing coverage of the 15 cases across regions was 516× (range: 121 to 989×) and over 94% of the targeted regions were covered by at least 100 reads. Variant calling was performed using 6 different variant callers, as previously described.25 (link) For each variant caller, low-quality variants were excluded using default thresholds and only those variants identified by >2 algorithms were considered. Bioinformatic pipeline included a filtering process to exclude intronic, synonymous, and single-nucleotide polymorphism variants, and to select driver mutations with potential functional effect (Supplementary Methods, Supplemental Digital Content 3, http://links.lww.com/PAS/B443 ).
9
Comprehensive Genomic Profiling of B-cell Lymphomas
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Twenty-seven PBL were examined for the mutational status of 94 B-cell lymphoma-related genes (Online Supplementary Table S2) using a SureSelectXT Target Enrichment System Capture NGS strategy library (Agilent Technologies, Santa Clara, CA, USA) before sequencing with MiSeq equipment (Illumina, San Diego, CA, USA). The bioinformatic pipeline included a filtering process excluding intronic, synonymous and single nucleotide polymorphic variants and a selection of driver mutations with potential functional effect (Online Supplementary Methods, Online Supplementary Figure S1). The cancer cell fraction carrying each specific mutation (CCFmut) was calculated as previously described.18 (link) As applied to copy number alterations, mutations were classified as clonal or subclonal if their cancer cell fractions were ≥85% or <85%, respectively.19 (link) Previously published mutational data from 28 BL, 203 PCM and 295 ABC-DLBCL were used for comparisons.24–26 (link)
10
Comprehensive CLL Driver Gene Mutational Profiling
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The mutational status of 28 CLL driver genes was examined using a deep-targeted NGS strategy in the 406 patients and 48 sequential samples. The genes were TP53, SF3B1, BIRC3, NOTCH1 and ATM, recently analyzed for the same cohort,26 (link) and 23 additional genes (POT1, NFKBIE, ZNF292, XPO1, EGR2, FBXW7, MGA, KLHL6, RPS15, MYD88, DDX3X, BRAF, NXF1, DTX1, BCOR, CCND2, KRAS, IRF4, MED12, ZMYM3, NRAS, TRAF3 and PIM1), which were selected among the most frequently mutated in prior whole-genome/exome sequencing studies (Supplementary Table S2 ).5 (link), 6 (link) Deep-targeted NGS libraries were performed using the Access Array system (Fluidigm, South San Francisco, CA, USA) (Supplementary Table S3 ) and/or the Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA) (Supplementary Table S4 ) before sequencing in a MiSeq equipment (Illumina) (Supplementary Methods ).
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