Miseq v2 instrument

Manufactured by Illumina

The MiSeq v2 instrument is a benchtop sequencing platform designed for targeted sequencing applications. It utilizes Illumina's proprietary sequencing-by-synthesis technology to generate high-quality sequencing data. The instrument can be used to sequence a variety of sample types, including DNA and RNA. The MiSeq v2 is capable of producing up to 15 gigabases of data per run.

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Lab products found in correlation

Nextera xt, by Illumina (1 mentions) Miseq desktop sequencer, by Illumina (1 mentions) Nextera xt index primer, by Illumina (1 mentions) Pacbio rs 2 sequencing technology, by Pacific Biosciences (1 mentions) Miseq sequencing, by Illumina (1 mentions) Nextera xt dna sample preparation kit, by Illumina (1 mentions) Genomic dna extraction kit, by Promega (1 mentions) Smrtbell template prep kit, by Pacific Biosciences (1 mentions) Truseq dna sample preparation protocol, by Illumina (1 mentions) Pacbio rs 2 sequencing platform, by Pacific Biosciences (1 mentions) Bluepippin size selection system, by Sage Science (1 mentions) Monarch genomic dna purification kit, by New England Biolabs (1 mentions) 50 ml conical tube, by Sarstedt (1 mentions)

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MiSeq v2 MiSeq instrument

4 protocols using miseq v2 instrument

Comprehensive BRCA and PALB2 Sequencing

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BRCA1, BRCA2 and PALB2 analysis was successfully conducted on 108 samples using Illumina’s Miseq desktop sequencer. Target enrichment was achieved by high throughput PCR. Primers were designed for the complete coding region including splice site regions of BRCA1 (31 amplicons), BRCA2 (42 amplicons) and PALB2 (19 amplicons) using Primer XL (www.pxlence.com). PCR conditions according to the protocol described by De Leeneer et al. were utilised [28 ].
Library preparation was performed using a modified version of the Nextera XT (Illumina) protocol. Sequencing was conducted on the MiSeq v2 instrument (Illumina Inc.) according to manufacturer’s instructions. The approach is described in detail by De Leeneer et al. [28 ].

Francies F.Z., Wainstein T., De Leeneer K., Cairns A., Murdoch M., Nietz S., Cubasch H., Poppe B., Van Maerken T., Crombez B., Coene I., Kerr R., Slabbert J.P., Vral A., Krause A., Baeyens A, & Claes K.B. (2015). BRCA1, BRCA2 and PALB2 mutations and CHEK2 c.1100delC in different South African ethnic groups diagnosed with premenopausal and/or triple negative breast cancer. BMC Cancer, 15, 912.

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Dual-platform Bacterial Genome Sequencing

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(i) Illumina MiSeq sequencing. Total DNA was extracted using a Qiagen genomic tip-100 (anion-exchange tip) (Qiagen, Valencia, CA) according to the kit manual, with minor modifications. Briefly, 6 ml of an overnight culture with vancomycin at 25 μg/ml, fusidic acid at 25 μg/ml, and rifampin at 50 μg/ml was used for each DNA sample. To break up the cells, 80 μl of 100 mg/ml lysozyme was used with 2 h of incubation at 37°C. The DNA samples were diluted to 0.3 ng/μl, and 5 μl was used for library generation using a Nextera XT DNA sample preparation kit and Nextera XT index primers (Illumina, San Diego, CA). Sufficient sample was diluted to 600 μl to provide a 15- to 20-pmol multiplexed library and sequenced on an Illumina MiSeq V2 instrument as 2×150 bp paired-end reads.
(ii) PacBio single-molecule sequencing. DNA was isolated as described above for Illumina sequencing, and 10 μg of high-quality DNA was used to make large insert libraries (10 kb) to be sequenced using the Pacific Biosciences (PacBio) RS II sequencing technology (Pacific Biosciences, Menlo Park, CA). For each sample, we used one PacBio RS II SMRT cell.

García-Solache M., Lebreton F., McLaughlin R.E., Whiteaker J.D., Gilmore M.S, & Rice L.B. (2016). Homologous Recombination within Large Chromosomal Regions Facilitates Acquisition of β-Lactam and Vancomycin Resistance in Enterococcus faecium. Antimicrobial Agents and Chemotherapy, 60(10), 5777-5786.

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Genomic DNA Extraction and Sequencing

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The harvested cells from the main culture were centrifuged and resuspended in 1 ml of fresh R5− medium. The resuspended cells were frozen with liquid nitrogen, and then lysed by grinding using mortar and pestle. The lysate was then centrifuged at 4°C, 3000 g for 10 min, and the supernatant was collected. The genomic DNA was prepared using genomic DNA extraction kit (Promega) as manufacturer's protocol. The extracted genomic DNA was used for construction of both PacBio genome sequencing library and Illumina short read sequencing library. For PacBio genome sequencing library, 5 μg of genomic DNA was used as input of the SMRTbell™ Template Prep Kit (Pacific Biosciences) and the library was constructed as manufacturer's protocol. After removing fragments smaller than 20 kb by the Blue Pippin Size selection system (Sage Science), the library was sequenced using the PacBio RS II sequencing platform (Pacific Biosciences). For Illumina short read sequencing library, the genomic DNA was fragmented to ∼200 bp using Covaris (Covaris Inc.) with the following condition; Power 175, Duty factor 20%; C. burst 20; Time 20 s; Cycle 8 times. Then, the library was constructed from fragmented genomic DNA by using TruSeq DNA Sample preparation protocol (Illumina). The library was sequenced on the MiSeq v2 instrument (Illumina) with 50 bp read recipe.

Hwang S., Lee N., Jeong Y., Lee Y., Kim W., Cho S., Palsson B.O, & Cho B.K. (2019). Primary transcriptome and translatome analysis determines transcriptional and translational regulatory elements encoded in the Streptomyces clavuligerus genome. Nucleic Acids Research, 47(12), 6114-6129.

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Genomic Analysis of P. aeruginosa Knockout Strains

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Glycerol stocks of P. aeruginosa WT and G2L4 RT knock-out strains were inoculated into 5-mL LB medium in a 50-mL conical tube (Sarstedt) and incubated at 37˚C for 16-18 h with shaking (200 rpm). The culture was then centrifuged at 4000 x g for 5 min, and genomic DNAs were extracted by using a Monarch Genomic DNA Purification Kit (New England Biolabs) according to the manufacturer's protocol. 1 µg of each genomic DNA was submitted to the Genome Sequencing and Analysis Facility (GSAF) at the University of Texas at Austin and sequencing libraries were prepared and sequenced on an Illumina MiSeq V2 instrument to obtain ~1 million 2
x 250 nt paired end reads per sample. Reads were mapped to the customized P. aeruginosa AZ-PAE12409 reference genome, which contains the targetron inserted at the designated location and the pBL1 vector used to express the targetron, using BWA with the default settings (Li and Durbin, 2010) . The genomic DNA coverage was calculated as mean coverage of 500-bp bins along the genomic sequences and plotted using R. Variants were called using freeBayes on bam files from genomic alignment of the WT or KO dataset, with the following settings: --ploidy 1 --min-mapping-quality 30 --min-alternate-count 10 ( Garrison and Marth, 2012) . The statistic test on KOspecific variants (point mutations) against the WT was analyzed by VarScan (Koboldt et al., 2009) .

Park S.K., Mohr G., Yao J., Russell R., & Lambowitz A.M. (2022). Group II Intron-Like Reverse Transcriptases Function in Double-Strand Break Repair by Microhomology-Mediated End Joining.

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