A virus (hCoV-19/Brazil/PE-IAM19/2020) containing a rare mutation N501Y in the spike protein was sent to Laboratory of Respiratory Viruses and Measles, the WHO Regional Reference Laboratory in Americas for SARS-CoV-2, for viral isolation and whole genome resequencing. The viral isolation was conducted using 200 µL of the clinical sample inoculated in VERO CCL-81 cells and after the third passage the cytopathic effect (CE) was observed. The supernatant was recovered, and the viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen). These experiments were performed at the BSL3 facility. The viral isolation was confirmed by the real time RT-PCR test targeting the gene E Kit Molecular Bio Manguinhos SARS-CoV-2 (E/RP). The RNA extracted from clinical sample and from isolated viruses from the third passage were submitted to whole genome sequencing using the RT and PCR multiplex protocol [12 (link)]. Amplified PCR products were purified using AMPure XP Beads (Beckman Coulter) following and quantified using the Qubit® dsDNA HS Assay Kits (Invitrogen) following the manufacturer’s instruction. The library was prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, CA, USA) and sequencing was performed in the Illumina MiSeq machine using MiSeq Reagent Kit v2 Micro employing a paired-end strategy.
Miseq reagent micro kit v2
Manufactured by Illumina
Sourced in United States, Brazil
The MiSeq Reagent Micro Kit v2 is a set of reagents designed for use with the Illumina MiSeq sequencing system. The kit provides the necessary materials for sample preparation and sequencing on the MiSeq platform.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (10 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (6 mentions)
Miseq system, by Illumina (5 mentions)
Nextera xt dna library preparation kit, by Illumina (4 mentions)
Miseq instrument, by Illumina (3 mentions)
Qubit double stranded dna dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Exosap it pcr product cleanup reagent, by Thermo Fisher Scientific (2 mentions)
Dna prep kit, by Illumina (2 mentions)
Miseq reagent kit v2, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Miseq reagent nano kit v2, by Illumina (2 mentions)
Easymag kit, by Thermo Fisher Scientific (2 mentions)
Covidseq test, by Illumina (2 mentions)
7500 fast real time pcr instrument, by Thermo Fisher Scientific (2 mentions)
Gridion, by Oxford Nanopore (2 mentions)
Nextera xt library prep kit, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Bioanalyzer high sensitivity dna assay, by Agilent Technologies (1 mentions)
S2 system, by Covaris (1 mentions)
38 protocols using miseq reagent micro kit v2
1
Viral Isolation and Genomic Sequencing of SARS-CoV-2 Variant
2
Targeted Sequencing of Amplicon Pools
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The DNA concentration of each amplicon pool was measured at the Qubit and adjusted to 1 ng µl−1. An aliquot of 130 µl was sheared in a Covaris S2 system to generate DNA fragments of ~300 bp in length. For the generation of fragment libraries, 50 µl of the sheared DNA solution was purified with 1.5 vol. Agencourt AMPureXP (A63881, Beckman Coulter). Twenty nanograms of purified DNA were used to prepare fragment libraries with the NEBNext Ultra II DNA Library Prep kit for Illumina (E7645L, New England Biolabs) and NEBNext unique dual index primer pairs (E6440L, New England Biolabs). For amplification, eight PCR cycles were applied. DNA fragment sizes of fragment libraries were monitored using the Bioanalyzer High Sensitivity DNA assay (5067–4626, Agilent Technologies). Equimolar amounts of individually barcoded libraries were pooled. The library pool was denatured with NaOH and finally diluted to 10 pM. Sequencing was performed on an Illumina MiSeq system (MiSeq Reagent kit v2 Micro; 2×151 bp paired end reads, 2×8 bp indices and 1 % control v3 PhiX) and generated between 113 687 and 269 991 read pairs for each of the 26 amplicon pools.
3
CRISPR Off-Target Analysis by Deep Sequencing
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Cas-OFFinder (http://www.rgenome.net/cas-offinder/ (accessed on 23 March 2021)) [27 (link)] was used to identify the potential off-targets that differed from sgIDH2_1 and sgIDH2_2 by up to three mismatches (Table 1 ). The samples used for the RFLP analyses were dually used as templates in a two-step PCR strategy to analyze the on-targets and 16 potential off-targets. In the first step, the PCR primers for each locus contained an adapter sequence. The amplicons were purified using AMPure Beads (BD Biosciences, Franklin Lakes, NJ, USA), and in the second step, they were re-amplified with primers containing an adapter sequence that overlapped the first primers and an index sequence in the reverse primers. The final PCR products underwent a second round of purification using the AMPure Beads prior to library construction. Following the manufacturer’s protocol, the purified PCR products were pooled in equimolar amounts and sequenced on an Illumina MiSeq instrument with a MiSeq Reagent Kit v2 Micro (500 cycles). The deep sequencing data were analyzed through CRISPResso2 (https://crispresso.pinellolab.partners.org/submission (accessed on 20 April 2022)) [28 (link)] to evaluate the editing efficiency and possible off-target effects using default parameters.
4
SARS-CoV-2 Genome Sequencing and Variant Detection
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Full genome sequencing was performed on a subset of SARS-CoV-2 positive specimens. Specimens with an E gene CT value over 30 were deemed too low in viral content to sequence and were usually excluded. The full genome was amplified as 1.2 kb amplicons using the Freed primer scheme27 (link). Library preparation and sequencing was carried out using either Oxford Nanopore Technologies (ONT) (Oxford, UK) or Illumina (San Diego, USA) methodologies. The ARTIC LoCost protocol28 and Ligation sequencing kit (SQK-LSK109) were used to prepare Nanopore libraries, which had 15–20 ng loaded onto the FLO-MIN 106D flow cells (ONT) for sequencing. Sequence data using the ONT protocol was compiled with the artic 1.1.3 pipeline29 . Illumina libraries were prepared with the DNA Prep Kit and sequenced on a MiSeq using the 300 cycle MiSeq Reagent Kit V2 Micro or on a MiniSeq using the MiniSeq Mid Output 300 cycle kit (Illumina). Sequence data using the Illumina protocol was processed with the OICR fork30 of the ncov2019-illumina-nf pipeline31 and with an updated version using freebayes as the variant caller32 . Sequence quality was assessed using ncov-qc33 . Lineages were assigned using pangolin34 (link) and mutations detected using nextclade35 .
5
CRISPR Screening of Hematopoietic Stem Cells
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Genomic DNA was isolated from HSPCs using Quick extraction buffer. The targeted amplicons library was prepared following Illumina’s recommendation using a two-step PCR protocol. Briefly, nested PCRs were performed on each DNA sample using the HiFi KAPA polymerase (Roche). Following Illumina barcoding (Nextera indices; Illumina), PCR samples were pooled, beads were purified and quantified using Qubit dsDNA BR (Thermo Fisher Scientific). The library was sequenced on an Illumina MiSeq instrument using Illumina MiSeq Reagent Kit v2 Micro (300 cycles) with 50% PhiX spike-in (Illumina). After demultiplexing, each sample was assessed for quality and analyzed using CRISPResso v2 (Clement et al., 2019 (link)). For each of the samples, we provided the HDRT, the reference sequence, and the guide sequence, and applied a minimum base quality of Phred 25. We used a custom R script to quantify each allele within a quantification window of four nucleotides including one silent mutation followed by the targeted amino acid.
6
Influenza A Whole-Genome Sequencing Protocol
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For influenza A virus whole-genome sequencing, 8 μL of the viral RNA was used for multisegmented reverse transcription-PCR (M-RT-PCR) using influenza A virus universal primers for the amplification of the eight gene segments of all influenza virus subtypes in a multiplex reaction (42 (link)). The generated amplicons were purified using the ExoSAP-IT PCR product cleanup reagent (Invitrogen) and quantified using a Qubit dsDNA (double-stranded DNA) HS assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocols. The cDNA library was constructed using the Nextera XT DNA library preparation kit (Illumina) and submitted to sequencing with the Illumina MiSeq system (Laboratory of Respiratory Viruses and Measles, Oswaldo Cruz Foundation, FIOCRUZ, Rio de Janeiro, Brazil) using MiSeq reagent kit v2 micro (300 cycles; Illumina) (43 (link)).
7
Mitochondrial 16S rDNA Barcode Sequencing
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A ~120 base pair fragment of the mitochondrial 16S rDNA gene was used as barcode region for species identification. Library preparation was carried out as described previously [28 (link)] with minor modifications. PCR products were indexed using the Illumina Nextera XT Index Kit v2 set A-D or the IDT-Illumina Nextera DNA UD Indexes Kit (Illumina, San Diego, CA, USA). Paired-end sequencing (2 × 150 bp) was performed with either the MiSeq Reagent Kit v2 or the MiSeq Reagent Kit v2 Micro (Illumina, San Diego, CA, USA) at a final loading concentration between 8–10 pM, depending on the instrument and the reagent kit, using the MiSeq system. PhiX DNA, added at a concentration of ~5%, served as sequencing control.
8
Influenza A Whole-Genome Sequencing Protocol
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For influenza A virus whole-genome sequencing, 8 μL of the viral RNA was used for multisegmented reverse transcription-PCR (M-RT-PCR) using influenza A virus universal primers for the amplification of the eight gene segments of all influenza virus subtypes in a multiplex reaction (42 (link)). The generated amplicons were purified using the ExoSAP-IT PCR product cleanup reagent (Invitrogen) and quantified using a Qubit dsDNA (double-stranded DNA) HS assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocols. The cDNA library was constructed using the Nextera XT DNA library preparation kit (Illumina) and submitted to sequencing with the Illumina MiSeq system (Laboratory of Respiratory Viruses and Measles, Oswaldo Cruz Foundation, FIOCRUZ, Rio de Janeiro, Brazil) using MiSeq reagent kit v2 micro (300 cycles; Illumina) (43 (link)).
9
CHIKV Genome Sequencing by Illumina
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Whole CHIKV genomes were determined by Illumina sequencing. Vero cell virus culture supernatant was harvested and then concentrated using Millipore centrifugal filter unit (100,000 kDa) for 20 min at 40,000× g (Merck Millipore Ltd., County Cork, Ireland). Total RNA was extracted from 150–200 μL of concentrated virus using the Roche High Pure RNA Isolation kit as per manufacturers’ instructions (Roche Diagnostic GmbH, Mannheim, Germany). DNA libraries were prepared with the Illumina TruSeq Stranded mRNA kit (Illumina Inc., San Diego, CA, USA). The DNA library was validated with a High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer (both from Agilent Technologies, Waldbronn, Germany). The concentration of the libraries was normalized to 4 nM using HT1 buffer and pooled together. The final concentration of the pooled DNA library was then re-examined using the Qubit dsDNA Assay kit (Invitrogen—Life Technologies, Carlsbad, CA, USA). The pooled DNA library was sequenced using a MiSeq Reagent Micro Kit, v2 (Illumina Inc., San Diego, CA, USA). The high throughput sequence reads from WGS were paired and used to construct contiguous sequences, de novo, using CLC Genomics Workbench 10.0 (Qiagen, Aarhus, Denmark) after quality assessment within FastQC Version 0.11 (Babraham Bioinformatics, Cambridge, UK).
10
SARS-CoV-2 Genome Sequencing and Lineage Analysis
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We observed an S gene dropout (i.e., gene not detected) in the sample SC2-9898 on May 2022 and then selected this sample for genome sequencing with the SARS-CoV-2 FLEX NGS panel (Paragon Genomics, Fremont, CA, USA) on the Illumina MiSeq platform. The library preparation was conducted according to the manufacturer’s protocol, and the sequencing was performed using the MiSeq Reagent Micro Kit v2 (Illumina Inc, San Diego, CA, USA). The FASTq files were obtained using the Local Run Manager Generate FASTQ Analysis Module v3.0 (Illumina Inc, San Diego, CA, USA) and submitted to the SOPHiA DDM v.5 platform. These files were analysed using the CleanPlex SARS hCoV2 pipeline for sequence alignment. Finally, the sequence was deposited in the GISAID database with the entry EPI_ISL_14381991.
For the EPI_ISL_12110384 and EPI_ISL_14284846 sequences, whole-genome sequencing was performed using the Illumina COVIDSeq protocol (Illumina Inc,, San Diego, CA, USA) on the Illumina sequencing platform. The pipeline ViralFlow was used to perform genome assembly, variant calling, and consensus generation [18 (link)]. To evaluate the quality and determine the lineage of the genome sequences, we analysed them on Nextclade Web version 2.3.1 [19 (link)].
For the EPI_ISL_12110384 and EPI_ISL_14284846 sequences, whole-genome sequencing was performed using the Illumina COVIDSeq protocol (Illumina Inc,, San Diego, CA, USA) on the Illumina sequencing platform. The pipeline ViralFlow was used to perform genome assembly, variant calling, and consensus generation [18 (link)]. To evaluate the quality and determine the lineage of the genome sequences, we analysed them on Nextclade Web version 2.3.1 [19 (link)].
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