The NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) was used to create the library for shotgun sequencing, according to the manufacturer’s instructions. To get fragments with an estimated length of 150–300 bp, samples were incubated with the enzyme mix contained in the kit at 37 °C for 15–20 min depending on the input-DNA-concentration (if DNA concentration was <100 ng, incubation time was 15 min; if DNA concentration was >100 ng, incubation time was 20 min). The NEBNext Multiplex Oligos for Illumina (Index Primers Set 1 and 2) (New England Biolabs, Ipswich, MA, USA) were used to create the final DNA libraries. To add adapters to the fragmented DNA, the number of PCR cycles was selected depending on the amount of input DNA as specified in the manufacturer’s protocol. Fragment sizes and quality of the final DNA libraries were evaluated using an Agilent Bioanalyzer with the Agilent DNA 1000 Reagent kit (both Agilent, Santa Clara, CA, USA). Finally, all samples were sequenced on an Illumina MiSeq platform, using the Illumina MiSeq v2 Reagent Kit (Illumina, San Diego, CA, USA).
Miseq reagent kit v2
Manufactured by Illumina
Sourced in United States, Canada, Germany, Switzerland, Denmark, Japan, Belgium, United Kingdom, China
The MiSeq Reagent Kit v2 is a laboratory equipment product designed for use with the Illumina MiSeq sequencing system. It provides the necessary reagents and consumables required to perform nucleic acid sequencing on the MiSeq platform.
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Lab products found in correlation
Miseq platform, by Illumina (175 mentions)
Miseq sequencer, by Illumina (69 mentions)
Miseq instrument, by Illumina (66 mentions)
Miseq system, by Illumina (53 mentions)
Nextera xt dna library preparation kit, by Illumina (36 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (27 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (26 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (25 mentions)
Nextera xt index kit, by Illumina (22 mentions)
Agencourt ampure xp bead, by Beckman Coulter (22 mentions)
Ampure xp bead, by Beckman Coulter (21 mentions)
Nextera xt dna sample preparation kit, by Illumina (20 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (19 mentions)
Miseq reagent kit v3, by Illumina (19 mentions)
2100 bioanalyzer, by Agilent Technologies (19 mentions)
Dneasy blood and tissue kit, by Qiagen (18 mentions)
Miseq desktop sequencer, by Illumina (15 mentions)
Nextera xt kit, by Illumina (15 mentions)
Kapa library quantification kit, by Roche (14 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (13 mentions)
785 protocols using miseq reagent kit v2
1
Shotgun Sequencing Library Preparation
2
Genomic and Plasmid DNA Isolation and Sequencing
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The isolation of genomic and plasmid DNA and sequence analysis was performed as described previously12 (link). Briefly, Campylobacter cells were transferred to MHB broth and grown for 72 h with vigorous shaking (175 rpm) at 42 °C. Cells were harvested after centrifugation and genomic DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA). Plasmids were isolated using the Qiagen plasmid midi-kit as recommended by the manufacturer. Library preparation for whole genome and plasmid sequencing was conducted using the Nextera XT sample preparation kit according to manufacturer’s instructions. Prepared libraries were sequenced in the Illumina Miseq platform with the Illumina MiSeq V2 reagent kit 2 × 150 cycles (Illumina Inc., CA). Sequence assembly was performed in the CLC Genomic Workbench v. 7.5.1 (Qiagen Inc.) Plasmid sequences were annotated using the RAST online tool (https://rast.nmpdr.org/rast.cgi )29 (link) and the NCBI Prokaryotic Genome Annotation Pipeline.
3
16S rDNA Microbiome Analysis Pipeline
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For microbiome analyses, fecal DNA samples were amplified by Illumina Miseq compatible primers, targeting the 16s rDNA V4–V5 region. Amplicons were purified by QIAquick Gel extraction kit (Qiagene, Madison, WI) and quantified by Qubit 2.0 Fluorometer (Invitrogen, Grand Island, NY) and Kapa SYBR fast qPCR kit (Kapa Biosystems, Inc., Woburn, MA). Equal amounts of amplicons were pooled with 10% of Phix control. Miseq v2 reagent kit (Illumina, Inc., San Diego, CA) was used to run the pooled samples on the Illumina Miseq machine. The Q score of this run was 86.59% and cluster density was 975±61. Data were analyzed, as previously described [18] (link). Primers used are found in Table S2 .
4
Illumina Sequencing of Deletion Mutants
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Libraries for Illumina sequencing were prepared with Nextera DNA Flex Library Prep kit (Illumina), using as input 500 ng of genomic DNA for each mutant strain. Sequencing was performed on an Illumina MiSeq sequencer using a MiSeq v2 reagent kit (300 cycles) in paired-end mode (2 × 150). In order to verify the expected genome edits in the deletion mutants, we performed de novo assemblies using the nf-core/bacass pipeline (59 (link)). The gene deletions in each strain were validated by inspecting the respective genomic regions in the assemblies.
5
16S rRNA Sequencing Pipeline using DADA2
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The V4 region of the 16s rRNA was amplified using barcoded dual index primers26 (link). Polymerase chain reactions were conducted and normalized using SequalPrep Normalization Plate Kit (Thermo Fisher Scientific, catalog no. A105100). The normalized reactions were then pooled and quantified using Kapa Biosystems Library qPCR MasterMix Quantification kit (catalog no. KK4873). Samples were sequenced on the Illumina MiSeq platform using the 500 cycle MiSeq V2 Reagent Kit (catalog no. MS-102-2003).
The FASTQ sequences were analyzed using the DADA2 method27 (link),28 (link). Amplicon sequence variants (ASV) were compiled using the DADA2 tutorial (https://benjjneb.github.io/dada2/tutorial.html ). Briefly, after assessing the quality of both the forward and reverse reads, the forward and reverse reads were filtered and trimmed. Error rates were determined, as well as the number of unique sequences per sample submission. The forward and reverse reads were then merged and chimeras removed. Taxonomy was assigned using the RDP training set.
The FASTQ sequences were analyzed using the DADA2 method27 (link),28 (link). Amplicon sequence variants (ASV) were compiled using the DADA2 tutorial (
6
Genomic DNA Sequencing and Annotation
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Sequencing libraries of genomic DNA were prepared and normalized with the Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA, United States) as recommended by the manufacturer. Sequencing of prepared libraries was conducted in the Illumina MiSeq platform using the Illumina MiSeq V2 Reagent kit and 2 × 250 cycles. Sequence assembly was conducted using the CLC Genomic Workbench v. 7.0 and the microbial genome finishing module. Identification, clustering and segregation of plasmid sequences from chromosomal sequences was performed with plasmidSPAdes (Antipov et al., 2016 (link)) and the PHASTER web server (Arndt et al., 2016 (link)). Several contigs were joined manually by consulting reference sequences. Assembled genomic and plasmid sequences were deposited in GenBank as listed in Table 1 . Sequences were annotated using the NCBI Prokaryotic Genome Annotation pipeline. RAST1 (Overbeek et al., 2014 (link)) and PATRIC v. 3.5.392 (Wattam et al., 2014 (link)) tools were used to annotate whole genomic sequences of S. aureus for comparative genomic analysis.
7
Optimizing Genome Recovery via Slow Ramp PCR
8
Evaluating SARS-CoV-2 PCR Enzyme Efficiency
9
Comparative Sequencing of Biological Samples
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Except for the four samples (2800M, components B and C of the 2391c standard reference material®, and M2) detected by both platforms in concordant studies, all other samples were sequenced on the MGISEQ-2000RS platform.
For MGISEQ-2000RS sequencing, libraries were prepared using the MGIEasy Amplicon Library Preparation Kit (MGI) as described in a previous publication [31 (link)], and sequenced using an MGISEQ-2000RS High-throughput Sequencing Kit (MGI) with a read length set at 350 bases. For Miseq FGx sequencing, libraries were prepared using the Truseq® DNA PCR-Free HT Kit (Illumina), and quantified using the KAPA Library Quantification Kit (Roche, Basel, Switzerland) on a 7500 realtime PCR system. The MiSeq v2 Reagent Kit (300 cycles PE; Illumina) was used for sequencing.
For MGISEQ-2000RS sequencing, libraries were prepared using the MGIEasy Amplicon Library Preparation Kit (MGI) as described in a previous publication [31 (link)], and sequenced using an MGISEQ-2000RS High-throughput Sequencing Kit (MGI) with a read length set at 350 bases. For Miseq FGx sequencing, libraries were prepared using the Truseq® DNA PCR-Free HT Kit (Illumina), and quantified using the KAPA Library Quantification Kit (Roche, Basel, Switzerland) on a 7500 realtime PCR system. The MiSeq v2 Reagent Kit (300 cycles PE; Illumina) was used for sequencing.
10
MicroRNA profiling of MDA-MB-231 cells with LOC550643 knockdown
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After the MDA-MB-231-IV2-1 cells were transfected with si-LOC550643 and the scrambled control for 48 h, the total RNA was extracted from two samples by using TRIZOL reagent. The small RNA library was prepared using the NEBNext small RNA library prep kit (New England Biolabs). The library preparation process is described in details in our previous study (Tseng et al., 2017 (link)). Finally, the small RNA profiles of the MDA-MB-231-IV2-control and MDA-MB-231-IV2-LOC550643 knockdown were performed using the MiSeq V2 reagent kit (150 cycles; Illumina, San Diego, CA, United States). The sequencing data were analyzed using our own tool (Pan et al., 2014 ). All microarray raw data were deposited in the NCBI GEO, and they are all accessible (accession number: GSE175514 ).
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