This protocol, similar to the LamPORE SARS-CoV-2 protocol, is used to test patient samples for the presence of the SARS-CoV-2 virus. However, this protocol is targeted at users who are working in lower-throughput facilities and do not have enough samples to fill up a 96-well plate at a time. Here, users are instructed to test 20 samples and 4 controls (two wells of Positive Control and two wells of No Template Control), each with an individual combination of barcodes, at a time. After the test is complete, the first library is flushed from the flow cell using the Flow Cell Recalibration Kit, and a second library with different barcode combinations can be loaded and tested.

The Flow Cell Recalibration Kit allows sequential runs of multiple LamPORE tests on the same flow cell. This procedure provides the opportunity to use the same flow cell several times, reducing the cost per test. Following the recalibration step, Storage Buffer can be introduced into the flow cell, allowing storage of the flow cell before subsequent library additions. Alternatively, the flow cell can be re-used immediately.

LamPORE has been developed by Oxford Nanopore Technologies to enable the simple and rapid detection of target regions of single or multiple genomes in a highly multiplexed manner.

In this kit, LamPORE is designed to detect the presence or absence of the SARS-CoV-2 virual RNA responsible for the COVID-19 pandemic in respiratory specimens (such as oropharyngeal and nasopharyngeal swabs) and saliva.

LamPORE combines barcoded multi-target isothermal amplification, 15-minute barcoded library preparation and real-time nanopore sequencing. By placing unique molecular barcodes in the Loop-mediated isothermal amplification (LAMP) reaction and coupling these with Oxford Nanopore's rapid barcoding adapters, a dual indexing approach is achieved, enabling a large number of barcode combinations to be generated and analysed.

The test deploys a simple workflow:

Loop-mediated Isothermal Amplification (LAMP) is a single-tube technique of targeted amplification which can generate micrograms of product from tens of copies of the target region. Reverse Transcription LAMP (RT-LAMP) combines LAMP with a reverse transcription step to allow the amplification and subsequent detection of RNA targets.

LamPORE deploys an RT-LAMP specific to three regions of the SARS-CoV-2 genome:

• N Gene
• E gene
• ORF1a gene

Additionally, a set of primers to amplify the human β-actin mRNA are included. The primers target either side of a splice junction and do not amplify from genomic DNA. As long as the sample has been collected and prepared correctly, β-actin RNA should be present in all the swab and saliva samples, regardless of their SARS-CoV-2 status, and so this provides a way to differentiate between true negatives and invalid samples.

To perform amplification, a strand-displacing polymerase is added to an RNA sample, primers and the reaction is incubated at 65°C for 35 minutes. In the initial stages of the reaction, the enzyme produces a series of dumbbell-shaped cDNA molecules and these are then exponentially amplified. Amplification of the dumbbell-shaped molecules results in long DNA strands consisting of concatenated copies of the original target regions.

To enable pooling of multiple samples into a single analysis run, the LamPORE SARS-CoV-2 primers include a 10-nucleotide molecular barcode on the Forward Inner Primer (FIP). Following amplification, several copies of this barcode are incorporated into each DNA concatemer.

At the end of the reaction, the enzymes are inactivated by incubation at 80°C for 5 minutes. LAMP produces a variety of products including multimeric DNA with inverted repeats. These complex amplification products are converted into nanopore sequencing libraries using the rapid barcoding chemistry.

Successful LAMP reactions are often inferred from a proxy measurement, such as increased turbidity or a colour change. However, although the LAMP reaction itself is very robust, these proxy measurements are less robust and can be affected by substances present in biological samples. It is also not uncommon to see a colour change or increase in turbidity in no-template controls, arising from amplification of primer artefacts, which would lead to a false positive call. Instead of relying on proxy measurements, sequencing can be used as a readout. On-target amplification events contain sequences that are not present in the primers and can be identified without ambiguity by alignment. In addition, sequencing provides an opportunity to amplify and detect multiple targets in a single tube.

To enable sequencing as a readout, a sequencing library must first be prepared. LamPORE uses the rapid barcoding library preparation method for three reasons: the first reason is speed and simplicity. Secondly, being transposase-based, the preparation method cuts the loop products generated during LAMP, turning them into linear molecules ready for sequencing. Finally, rapid barcoding chemistry incorporates a barcode in the sequencing adapter enabling a combinatorial barcode approach which delivers a far higher capacity for multiplexing samples into an analysis run.

To prepare a rapid barcoded library, you will need to:

• Tagment your DNA using the rapid barcodes; this cuts the DNA and simultaneously attaches a pair of barcodes to the fragments
• Pool the barcoded samples
• Purify the sample using SPRI beads
• Attach sequencing adapters to the DNA ends
• Prime the flow cell, and load your DNA library into the flow cell

Per-sample results of the assay are returned as positive, negative, inconclusive, or invalid.

During nanopore sequencing, an electrical current is measured as strands pass through each pore on the flow cell. Conversion of this current into basecalls can start while a strand is translocating.

LamPORE reads

A LamPORE reaction creates an end product with multiple copies of the barcoded target region and a sequencing barcode at the start of each read.

Basecalling

Raw signal data processing basecalling algorithms are contained in the MinKNOW software.

The data at this point is deposited in files according to the barcode on the rapid sequencing chemistry with all files containing rapid barcode 1 in folder 1 and all files containing barcode 2 in folder 2 etc.

At the end of the experiment, when all reads have been basecalled and placed in appropriate folders, MinKNOW initiates the downstream analysis pipeline.

Analysis pipeline

The LamPORE analysis pipeline:

• Demultiplexes the reads by the FIP barcodes
• Aligns the reads against the SARS-CoV-2 genomic targets and human β-actin
• Generates a TSV file describing the number of reads for each barcode which align to the targets.

A PDF report is also generated, containing classification results for every barcoded sample.

Analysis pipeline in detail

The first step of the enalysis pipeline is to classify reads containing the LAMP barcodes. After this step is performed, the classified reads move onto an alignment step.

The LamPORE primers are designed such that they leave a gap where genuine SARS-CoV-2 sequence is replicated by the LAMP reaction in the case of a positive sample.

All reads are aligned against the SARS-CoV-2 genome and only reads with sequence information in that region are called positive.

Distinguishing between valid reads and primer artefacts by alignment works by: a) valid reads consist of repeats that align across the majority of the target region, whereas b) primer artefacts align as short segments interspersed with gaps.

By pairing LAMP with nanopore sequencing, it is easy for a genuine positive to be differentiated from amplification artefacts. This enables the assay to be highly specific as reads containing the necessary sequence serve as an unambiguous identification for the presence of the target.

The determination of the sample result applies the following rules:

• Positive: SARS-CoV-2 detected (≥50 SARS-CoV-2 reads)
• Inconclusive: The test should be repeated (20 ≤ SARS-CoV-2 reads ≤ 49)
• Negative: SARS-CoV-2 not detected (<20 SARS-CoV-2 reads)
• Invalid: insufficient number (<50) of classified reads from both SARS-CoV-2 and β-actin to make a call; the test should be repeated

It is important to note that this read-out is to be used by a healthcare professional in combination with patient symptoms and healthcare history to inform the next course of treatment.

The LamPORE COVID-19 Test Kit 96 Plex S, the SARS-CoV-2 Control kit S and the Flow Cell Recalibration Kit have a shelf life of 3 months when stored at -20°C.

The Positive Control (CTL) tubes should be taken out of the kit and stored at -80°C for up to 3 months.

We do not reccomend repeated freeze-thaw of the Positive Control (CTL) and as such, sufficient aliquots are provided in the kit to minimise this.

The GridION device contains all the hardware required to control up to five flow cells and acquire the data. The device is further enhanced with high performance GPU technology for real-time basecalling. Read more in the GridION IT requirements document.

MinKNOW

The MinKNOW software (OND 19.12) controls the nanopore sequencing device, collects sequencing data in real time and processes it into basecalls. MinKNOW can also demultiplex reads by barcode.

Analysis

At the end of the sequencing experiment, when all reads have been basecalled, MinKNOW initiates the downstream analysis pipeline.

1. A green tick - The flow cell is within warranty and can be used for a LamPORE test.

2. A yellow exclamation mark - The flow cell is below warranty and should not be used for the LamPORE test. Please contact support@nanoporetech.com to arrange a replacement.

Note: The indicator of quality (exclamation mark or tick) will only remain visible during a MinKNOW session. Once the MinKNOW service has ended, the status of the flow cell will be erased.

This method uses a combinatorial barcoding approach. One set of barcodes is contained in the FIP primers used in the LAMP reactions, and an additional set of Rapid Barcodes is also supplied. All samples and controls in a given row of the plate will receive the same FIP barcode, and all samples and controls in a given column of the plate will receive the same Rapid Barcode, as shown in the figure below.

Here, samples in row A of the plate receive FIP barcode 01, samples in row B receive barcode FIP 02 etc. Meanwhile, samples in column 1 receive the Rapid Barcode 01, samples in column 2 receive the Rapid Barcode 02 etc. This way, each sample will receive a unique combination of FIP barcode and Rapid Barcode.

It is necessary to include at least two wells of No Template Control and at least two wells of Positive Control per plate, which leaves a maximum of 92 samples per plate. If you are running fewer than 92 samples at a time, we recommend filling up columns rather than rows with samples. Any unused wells in a column should be filled with LAMP Master Mix/LAMP Primers and 20 μl nuclease-free water and processed in the same way as a sample.

The files with test results are placed in the /data folder, with the following structure/file naming convention:

{data}/
  {experiment_id}/
    {sample_id}/
      {date_time}_{device_id}_{flowcell_id}_{protocol_unique_identifier}/

e.g. `/data/lampore_20200617/lampore/20200617_1636_X1_FAO39500_e29f6fcd`

The experiment results can be found in the lamPORE_report PDF and the lamPORE_results TSV file.

The report is a TSV file describing number of reads which align to given targets and have given barcodes, as well as metadata for run tracking. A PDF report is also generated, containing results for every barcoded sample.

The fields in the TSV file are as follows:

Column	Meaning
Barcode	RB and FIP barcode combination
target_human_ACTB	Number of reads aligning to the human β-actin target
target_nCoV2019_AS1	Number of reads aligning to the ORF1a target in the SARS-CoV-2 genome
target_nCoV2019_E1	Number of reads aligning to the E1 target in the SARS-CoV-2 genome
target_nCoV2019_N2	Number of reads aligning to the N2 target in the SARS-CoV-2 genome
target_unclassified	Number of unclassified reads (reads with barcodes that do not align with the correct specificity)
acquisition_run_id	A unique alphanumeric number to identify the run
protocol_group_id	Experiment name assigned when setting up the run in MinKNOW
sample_id	Name assigned to the flow cell when setting up the run in MinKNOW
flow_cell_id	Unique identification code of the flow cell
started	Experiment start date and time
call	Positive: SARS-CoV-2 detected (≥50 SARS-CoV-2 reads) Inconclusive: The test should be repeated (20 ≤ SARS-CoV-2 reads ≤ 49) Negative: SARS-CoV-2 not detected (<20 SARS-CoV-2 reads) Invalid: insufficient number (<50) of classified reads from both SARS-CoV-2 and β-actin to make a call; the test should be repeated

An example PDF report from the analysis pipeline and example TSV file are shown below.

During the RT-LAMP and library preparation, two controls from the kit are included: CTL (positive control), and NTC (no template control).

When preparing your samples, make a note of the positions of the CTL and NTC wells in each plate. Check the results in the analysis reports (found in the lamPORE_results TSV file and lamPORE_report PDF file) for these positions:

• NTC contains no SARS-CoV-2 RNA, and should give an Invalid result in the call column of the TSV file or PDF report. If it shows a Positive, Negative or Inconclusive result, this means that all tests in this plate are invalid and need to be re-run.
• CTL contains SARS-CoV-2 RNA, and is expected to give a Positive result in the call column of the TSV file or PDF report. If it shows a Negative, Inconclusive or Invalid result, this means that all tests in this plate are invalid and need to be re-run.

• The aim is to remove most of the initial library and prepare the flow cell for the loading of a subsequent library
• The Flow Cell Recalibration Kit contains all solutions required for removal of the initial library
• The experiment in MinKNOW should be allowed to end before recalibrating the flow cell
• After the flow cell has been recalibrated, a new library can be loaded or the flow cell can be stored at 4°C