Tonight I watched the November 13, 2018 recording of the “Sample barcoding: Sequencing in multiplex and Demultiplexing” Knowledge Exchange session in the Nanopore Learning Lesson Library. Julia Eales, an Application Scientist with Oxford Nanopore Technologies and Philipp Rescheneder presented. Eales spoke about the uses of multiplexing. I didn’t know that with dual-barcoding you can multiplex 2,304 samples. Barcoding reduces the per-sample cost of sequencing. Barcoding is also useful for time course experiments. There are PCR Barcoding and Native Barcoding kits. PCR Barcoding kits are useful when you have low starting amount of DNA and want to maximize throughput. The PCR barcoding expansion kit can be used in conjunction with the ligation sequencing kits. There are 12 and 96 barcode sets. There is also a stand-alone PCR barcoding kit that I have used. The PCR barcoding kits use rapid attachment of adapters. There are several ways of PCR barcoding with locus-specific tags. These methods often require specific sequences. Native barcoding is PCR-free. These kits are useful for sequencing native DNA and avoiding fragmentation. Eales also noted that native barcoding reduces time. An adapter is added by ligation and the NEB Quick Ligase. Rapid PCR barcoding is useful for lower DNA input and tolerance of shorter reads, since this kit uses the transposase system. The rapid barcoding kit uses barcoded rapid adapters attached in a single step. Eales noted that without cleanups the process takes about ten minutes. Eales explained that the PCR barcoding kit and native barcoding kits can be combined for dual barcoding! The New York applications group used dual barcoding preparing samples from colonies from swabs. They were able to sequence numerous samples this way in a single run. Eales ended by describing the flow cell wash procedure and how it was used to sequence lambda libraries prepared with individual native barcodes and sequenced for one hour before washing. There was little carry over between washes. Rescheneder explained how demultiplexing works. They noted that there are several tools for demultiplexing, including EPI2ME. Rescheneder recommends reviewing the number of unclassified reads. QCAT can be used with the command-line. They shared and compared the output of demultiplexing with these tools. I learned about some command-line tools I can use!
