Nanopore direct cDNA sequencing (SQK-DCS109) was performed using the flow cell R9.4 on the MinION machine (Oxford Nanopore Technologies) following the manufacturers’ instructions with minor modifications. Briefly, first strand cDNA was made from 100 ng of poly-A-enriched RNA using the VN primer and SuperScript II (Invitrogen) at 42°C for 50 min. After removal of RNA by RNase Cocktail Enzyme Mix (Thermo Fisher), second strand cDNA was made using random hexamers (Invitrogen) and LongAmp Taq Master Mix (New England Biolabs), which was followed by the End-prep and Adapter ligation before subjecting the library to the flow cell. The base-calling algorithm Albacore 2.3.1 (Oxford Nanopore Technologies) was used to process the raw FAST5 files. About half a million reads that passed the default quality threshold were mapped to the human genome (hg38 assembly) using Minimap2 (36 (link)) with -ax splice and -k14 options. Alignments with MAPQ < 20 were skipped. SAM files were converted to the BAM format using samtools 1.9 and visualized in the UCSC genome browser (33 (link)). Further data processing and analysis was conducted using the R software (version 3.4.3).
Albacore 2
Manufactured by Oxford Nanopore
Albacore 2.3.1 is a software package developed by Oxford Nanopore Technologies. It is designed to process and analyze data generated by Oxford Nanopore's DNA/RNA sequencing devices.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
Rnase cocktail enzyme mix, by Thermo Fisher Scientific (1 mentions)
Longamp taq master mix, by New England Biolabs (1 mentions)
Nextera xt library preparation kit, by Illumina (1 mentions)
Sqk lsk109, by Oxford Nanopore (1 mentions)
Minion device, by Oxford Nanopore (1 mentions)
Exp nbd103, by Oxford Nanopore (1 mentions)
2 protocols using albacore 2
1
Nanopore Direct cDNA Sequencing Protocol
2
Multi-platform Genomic Sequencing of Crustaceans
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DNA sequencing libraries were prepared for Illumina sequencing using the Nextera XT library preparation kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina MiSeq using V3 chemistry (Illumina; 2 × 150 bp for C. crangon (5 individual samples pooled) and 2 × 300 bp for C. maenas (single sample)). A Nanopore sequencing library was constructed using the ligation sequencing kit SQK-LSK109 and the native barcoding kit (EXP-NBD103) for 5 C. Crangon samples (Oxford Nanopore Technologies Ltd., Oxford, UK). The barcoded DNA samples were pooled in equimolar concentrations and the prepared library loaded onto a SpotON flowcell R9.4.1 (FLO-MIN106) and sequenced for 11 h on a MinION device (Oxford Nanopore Technologies). Data were base-called and demultiplexed locally on a laptop using Albacore 2.3.1 (base-calling software released by Oxford Nanopore Technologies).
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