This might seem like an obvious point, but because the Midnight amplicons are longer than the standard Artic amplicons and we have the existence of fragmented amplicons, we need to adjust the read length cut-offs used to filter reads. The SOP can be found here.īecause of the differences between the original ARTIC method and Midnight, amendments were made to the underlying assumptions in the ARTIC FieldBioinformatics package used to analyse data generated by tiled amplicon sequencing of SARS-CoV-2 and so our wf-artic Nextflow workflow was born. The ARTIC bioinformatics analysis workflow is globally recognised as the gold standard for the processing of ARTIC tiled amplicon SARS-CoV-2 genomes. Midnight however uses the rapid library preparation chemistry, improving turnaround time but due to the transposase tagmentation employed in this method read lengths are less than or equal to the intact amplicon length.ġ. ARTIC original used the ONT ligation sequencing kit and therefore all reads produced are equivalent to the amplicon length. In addition to the amplicon length the key difference between the Midnight protocol and the original ARTIC protocol with Nanopore sequencing is the library preparation method. These primers were designed using primalscheme by Quick et. The amended Midnight protocol uses longer 1200bp amplicons first proposed in a publication by Freed et.
#MIDNIGHT PROTOCOL FULL#
This enables full genome coverage even in situations where viral RNA might be more degraded or is present at low number of copies.
This protocol relies on 98 overlapping ~400bp amplicons split into two pools. There are many methods to sequence the genome of SARS-CoV-2 but one of the most popular remains the ARTIC Network protocol. First the rapid publication of the original sequence from Wuhan to ongoing surveillance to identify and track the emergence of variants of the virus that could be a public health concern. Sequencing of SARS-CoV-2 has been pivotal in the ongoing pandemic. Discuss lineage and clade assignment and how we keep up to date with new versions of these.
#MIDNIGHT PROTOCOL CODE#
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