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Barcode design
The Oxford Nanopore Technologies barcoding kits can place barcodes at the beginning and for some kits, also at the end of the strands for multiplexing several different samples in one sequencing experiment. The barcodes will reside in a kit-specific context sequence, and different kits will have different lengths of sequence before and after the barcode. However, the sequences of the barcodes themselves are identical, regardless of kit.
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The regions of a barcode
A complete barcode arrangement comprises three sections:
- The upstream flanking region, which comes between the barcode and the sequencing adapter.
- The barcode sequence.
- The downstream flanking region, which comes between the barcode and the sample sequence.
The barcode sequences remain constant across almost all of Oxford Nanopore Technologies' kits. For example, the flanking regions for barcode 10 in the Rapid Barcoding Kit (SQK-RBK114.24) are different from the flanking regions for barcode 10 in the Rapid PCR Barcoding Kit (SQK-RPB114.24), but the barcode sequence itself is the same. The exception is Native Barcoding kits, where the barcodes are the reverse complement of the standard barcodes.
Barcode and barcode flanking sequences can be found in the Chemistry technical document in the Nanopore Community.
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Barcode demultiplexing options
After a barcoded sequencing run has completed, the reads can be split into folders by barcode, using one of Oxford Nanopore's demultiplexing tools:
- Real-time barcode demultiplexing in MinKNOW
- Post-run barcode demultiplexing in MinKNOW
- Barcode demultiplexing in the Dorado basecall server
A brief description of the options is provided below.
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Barcode demultiplexing in MinKNOW
MinKNOW currently uses Dorado for both basecalling and barcode demultiplexing. It performs barcode demultiplexing in real-time, as the sequencing run progresses. MinKNOW demultiplexing is also available as a post-run analysis option.
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Barcode demultiplexing in Dorado
The barcoding algorithm in the Dorado basecall server uses a modified Needleman-Wunsch method. Each barcode is aligned to a section of the basecall, with a score assigned to each base in the sequence depending on whether the base was a match, mis-match or a gap. The combined scores for each barcode alignment are compared, and the barcode with the highest score is chosen as long as the score is above the defined threshold. The barcode sequences can be trimmed from the reads, as a command-line option.