For sequencing of virus stocks, the viral RNA was extracted from supernatants using the RNeasy Mini kit (Qiagen) and reverse-transcribed to cDNA with LunaScript RT SuperMix kit (New England Biolabs). Primer pools (Supplementary Table 1 ) targeting SARS-CoV-2 were designed using PrimalScheme tool45 (link) and PCR was done with PhusionFlash PCR master mix (Thermo Scientific). Sequencing libraries were prepared with NEBNext ultra II FS DNA library kit (New England Biolabs) according to the manufacturer’s instructions and sequenced using Illumina Miseq with v3 sequencing kit. Raw sequence reads were trimmed, and low quality (quality score <30) and short (<25 nt) sequences were removed using Trimmomatic version 0.3646 (link). The trimmed sequence reads were assembled to the reference sequence (NC_045512.2) using BWA-MEM47 algorithm implemented in SAMTools version 1.848 (link). Sequences of four SARS-CoV-2 isolates used in this study were deposited in GenBank: FIN-25 (GenBank MW717675), SR121 (GenBank MW717676), 85HEL (GenBank MW717677), and HEL-12-102 (GenBank MW717678).
Nebnext ultra 2 fs dna library kit
Manufactured by New England Biolabs
The NEBNext Ultra II FS DNA Library Kit is a reagent kit used for the preparation of DNA libraries for next-generation sequencing. The kit includes reagents and enzymes necessary for DNA fragmentation, end-repair, adaptor ligation, and library amplification.
Automatically generated - may contain errors
Lab products found in correlation
Phusion flash pcr master mix, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Lunascript rt supermix kit, by New England Biolabs (3 mentions)
Nebnext multiplex oligos for unique dual index kit, by Illumina (2 mentions)
R9.4.1 flow cell, by Oxford Nanopore (2 mentions)
Sqk lsk109 library preparation kit, by Oxford Nanopore (2 mentions)
Megaruptor, by Diagenode (2 mentions)
Wizard dna extraction kit, by Promega (1 mentions)
Hiseq x10 instrument, by Illumina (1 mentions)
Smart seq v4, by Takara Bio (1 mentions)
Novaseq platform, by Illumina (1 mentions)
7 protocols using nebnext ultra 2 fs dna library kit
1
SARS-CoV-2 Genome Sequencing Protocol
2
SARS-CoV-2 Genome Sequencing Protocol
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Viral RNA extracted from original swab samples and virus culture using the RNeasy minikit (Qiagen) was reverse transcribed to cDNA using a LunaScript RT SuperMix kit (New England Biolabs). Primer pools targeting SARS-CoV-2 were designed using the PrimalScheme tool (66 (link)), and PCR was conducted using PhusionFlash PCR master mix (Themo Fisher). Sequencing libraries were prepared using an NEBNext Ultra II FS DNA library kit (New England Biolabs) according to the manufacturer’s instructions and were sequenced using an Illumina Miseq with a v3 sequencing kit. Raw sequence reads were trimmed, and low quality (quality score of <30) and short (<25 nt) sequences were removed using Trimmomatic (67 (link)). The trimmed sequence reads were assembled to the reference sequence (NC_045512.2 ) using the BWA-MEM (68 ) algorithm implemented in SAMTools version 1.8 (69 (link)). Mutation frequencies in virus populations were estimated based on minority variant calling with LoFreq (70 (link)).
3
Genomic DNA Extraction and Sequencing
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Genomic DNA was extracted following the protocol outlined in a previous report [21 (link)]. Briefly, the Wizard DNA extraction kit (Promega) was used in the extraction of genomic DNA of all isolates. A dsDNA broad range quantification assay was used in the quantification of DNA extracts (Invitrogen). DNA libraries were prepared and sequenced using the NEBNext Ultra II FS DNA library kit (New England Biolabs) and HiSeq X10 instrument (Illumina), respectively.
4
SARS-CoV-2 Genome Sequencing Protocol
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Viral RNA extracted from original swab samples and virus culture using the RNeasy minikit (Qiagen) was reverse transcribed to cDNA using a LunaScript RT SuperMix kit (New England Biolabs). Primer pools targeting SARS-CoV-2 were designed using the PrimalScheme tool (66 (link)), and PCR was conducted using PhusionFlash PCR master mix (Themo Fisher). Sequencing libraries were prepared using an NEBNext Ultra II FS DNA library kit (New England Biolabs) according to the manufacturer’s instructions and were sequenced using an Illumina Miseq with a v3 sequencing kit. Raw sequence reads were trimmed, and low quality (quality score of <30) and short (<25 nt) sequences were removed using Trimmomatic (67 (link)). The trimmed sequence reads were assembled to the reference sequence (NC_045512.2 ) using the BWA-MEM (68 ) algorithm implemented in SAMTools version 1.8 (69 (link)). Mutation frequencies in virus populations were estimated based on minority variant calling with LoFreq (70 (link)).
5
RNA-seq Library Preparation for Freshly Sorted Cells
6
Multiplatform Sequencing of HMW DNA
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We first generated Oxford Nanopore long reads in-house on the MinION device using the SQK-LSK109 library preparation kit followed by sequencing on a R9.4.1 flow cell per the manufacturer’s instructions (Oxford Nanopore Technologies). After recovering less than 3 Gb of sequence data, we sent our remaining two HMW extracts to the UC Davis Core Lab to run on two PromethION flow cells. For the first PromethION run, a single library was prepared, loaded, and run for 48 hr. In an attempt to recover more sequence data, for the second PromethION flow cell, the HMW DNA was sheared using a Megaruptor (Diagenode, Inc.) set to a 50 kb target prior to a double library preparation allowing for a nuclease flush and a fresh library reload at the 24 hr mark of a 48 hr run. To generate the Illumina sequencing library, we used the NEBNext Ultra II FS DNA library kit (New England Biolabs, Inc); the initial enzymatic shearing step was accomplished via 10 min of incubation at 37°, after which we followed the manufacturer’s instructions. The library was indexed using NEBNext Multiplex Oligos for Illumina unique dual index kit (New England Biolabs, Inc). This library was run on one lane of a NovaSeq S-Prime flow cell at the Oklahoma Medical Research Foundation Clinical Genomics Center.
7
Long-Read Sequencing and Illumina Library Prep
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We first generated Oxford Nanopore long reads in house on the MinION device using the SQK-LSK109 library preparation kit followed by sequencing on a R9.4.1 flow cell per the manufacturer's instructions (Oxford Nanopore Technologies). After observing low yield, we sent our remaining two HMW extracts to the UC Davis Core Lab to run on two respective PromethION flow cells. For the first PromethION run a single library was prepared, loaded, and run for 48 hours. After limited yield in that run, for the second PromethION flow cell, the HMW DNA was sheared using a Megaruptor (Diagenode, Inc.) set to a 50 kb target prior to library preparation. Two libraries were made such that the flow cell ran for 24 hours, was flushed with a nuclease wash, and a second library was loaded for the final 24 hours of run time. To generate the Illumina sequencing library, we used the NEBNext Ultra II FS DNA library kit (New England Biolabs, Inc); the initial enzymatic shearing step was accomplished via 10 minutes of incubation at 37ºC, after which we followed the manufacturer's instructions. The library was indexed using NEBNext Multiplex Oligos for Illumina unique dual index kit (New England Biolabs, Inc). This library was run on one lane of a NovaSeq S-Prime flow cell at the Oklahoma Medical Research Foundation Clinical Genomics Center.
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