Feb 07, 2024
Long-read sequencing and data processing
- 1University of Oxford
Protocol Citation: Peter Kilfeather 2024. Long-read sequencing and data processing. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x5pklqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 07, 2024
Last Modified: February 07, 2024
Protocol Integer ID: 94800
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-020370
Monument Trust Discovery Award from Parkinson’s UK
Grant ID: J-1403
Abstract
Long-read sequencing and data processing methods from Kilfeather, Khoo et al., 2024
Protocol
Protocol
Twelve TRAP samples and three TOTAL samples were sequenced using the Oxford Nanopore Technologies MinION platform. TRAP samples were equally divided by age and genotype (N = 3 per age:genotype). Library preparation was performed using the cDNA-PCR kit (SQK-PCS109). Raw fast5 data was basecalled and demultiplexed using Guppy (v4.5.2). Read data from FASTQ
files were aligned to the mm10 genome (Gencode M25 GRCm38.p6) using minimap2 (v2.18, RRID:SCR_018550)57. Transcript level quantification was then performed using Salmon (v1.4.0, RRID:SCR_017036)58.