Approximately 1μg of DNA was used to generate a sequencing library according to the protocol provided for the SQK-LSK109 library preparation kit from Oxford Nanopore. After the DNA repair and end prep and adapter ligation steps, SPRIselect bead suspension (Beckman Coulter Cat No. B23318) was used to remove short fragments and free adapters. Qubit dsDNA assay was used to quantify DNA and approximately 300-400 ng of DNA library was loaded onto a MinION flow cell. Base calling was performed using albacore with the default filtering setting of Qscore >7 (de Lannoy et al., 2017 ) (BioProject Number PRJNA622927, BioSample SAMN14582895).
Sqk lsk109 library preparation kit
Manufactured by Oxford Nanopore
Sourced in United Kingdom
The SQK-LSK109 library preparation kit is a product offered by Oxford Nanopore. It is used for preparing DNA samples for sequencing on Oxford Nanopore's sequencing platforms. The kit includes the necessary reagents and protocols to convert DNA samples into a format suitable for loading onto Oxford Nanopore sequencing devices.
Automatically generated - may contain errors
Lab products found in correlation
Spriselect bead suspension, by Beckman Coulter (2 mentions)
R9.4.1 flow cell, by Oxford Nanopore (2 mentions)
Nebnext multiplex oligos for unique dual index kit, by Illumina (2 mentions)
Nebnext ultra 2 fs dna library kit, by New England Biolabs (2 mentions)
Megaruptor, by Diagenode (2 mentions)
Qubit dsdna assay, by Thermo Fisher Scientific (1 mentions)
5 protocols using sqk lsk109 library preparation kit
1
Nanopore Sequencing Library Preparation
2
Nanopore Sequencing Library Preparation
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Approximately 1 μg of DNA was used to generate a sequencing library according to the protocol provided for the SQK-LSK109 library preparation kit from Oxford Nanopore (Oxford Nanopore Technologies, Oxford Science Park, UK). After the DNA repair, end prep, and adapter ligation steps, SPRIselect bead suspension (Cat No. B23318, Beckman Coulter Life Sciences, Indianapolis, IN, USA) was used to remove short fragments and free adapters. Qubit dsDNA assay (ThermoFisher Scientific, Waltham, MA, USA) was used to quantify DNA and ∼300–400 ng of DNA library was loaded onto a MinION flow cell (MinION, RRID:SCR_017985 ) (BioProject: PRJNA634549, SRA: SRX8462258, SRX8462259).
3
Oxford Nanopore Sequencing Protocol
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Oxford Nanopore library preparation and sequencing was performed at JHSPH using a starting input amount of 350–1300 ng in 48 μL volume. Libraries were prepared following the SQK-LSK109 library preparation kit from Oxford Nanopore Technologies, with minor modifications as described in Ekeng et al. 25 The final pooled library was eluted in 15 μL Elution Buffer and 190 ng was diluted to 12 μL for loading onto the MinION flow cell. The MinION was run for a total of 48 hours per run and the resulting data was basecalled using Guppy version 3.0.3 with model dna_r9.4.1_450bps_fast.cfg. Adapter removal and demultiplexing was performed with Porechop 37 (link) and reads were filtered using Filtlong 38 with the following options: ‘--keep_percent 90 --target_bases 800000000.’
4
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.
5
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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