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 viral RNA, as well as five other targets in a respiratory panel, in respiratory specimens (such as oropharyngeal and nasopharyngeal swabs).

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 respiratory panel deploys an RT-LAMP specific to three regions of the SARS-CoV2 genome (N gene, E gene and ORF1a gene), the hemagluttinin gene of two strains of FluA (H1N1 and H3N2), the neuraminidase gene of FluB, the matrix protein gene of RSVA, and the nucleocapsid protein gene of RSVB.

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 or other respiratory panel targets 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 60 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 respiratory panel 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 targets of the respiratory panel and human β-actin
• Generates a TSV file describing the number of reads for each barcode which align to the targets.

Analysis pipeline in detail

The first step of the analysis 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 and other respiratory panel target sequences are replicated by the LAMP reaction in the case of a positive sample.

All reads are aligned against the SARS-CoV-2 genome and other respiratory panel targets and only reads with sequence information in those regions 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 or other target detected (≥20 reads for the viral target, <20 reads for other viral targets)
• Negative: SARS-CoV-2 and other targets not detected (<20 reads for all viral targets, ≥20 reads for β-actin)
• Invalid: insufficient number (<20) of classified reads from SARS-CoV-2, other respiratory panel targets, 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 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 20.12) controls the nanopore sequencing device, collects sequencing data in real-time and processes it into basecalls. MinKNOW can also demultiplex reads by barcode.

Analysis

During the experiment, MinKNOW initiates the downstream analysis pipeline and the analysis is performed.

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 barcode is contained in the FIP primers used in the LAMP reactions, and an additional set of 96 Rapid Barcodes is also supplied. All samples and controls in the plate will receive the same FIP barcode, and each sample and control in the plate will receive a unique Rapid Barcode. 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 each Positive Control per plate, which leaves a maximum of 88 samples per plate.

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.

The fields in the TSV file are as follows:

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 barcode_alignment TSV file) for these positions:

• NTC contains no SARS-CoV-2 RNA and should yield a very low count for any of the viral targets (<20) observed in the barcode_alignment TSV file. If the value exceeds 20, then this plate is invalid and needs to be re-run.
• Positive Controls contains SARS-CoV-2 or other respiratory panel target RNA and should yield a high count for any of the SARS-CoV-2 targets (>20) observed in the barcode_alignment TSV file. If the value is below 20, then this plate is invalid and needs to be re-run.