Joshua Quick from the University of Birmingham in the UK has developed several Nanopore protocols and tools. Tonight I watched Quick present at London Calling 2019 on “The ‘Three Peaks Challenge’ and developing extraction methods suitable for long-read, ultra-deep stool metagenomics on the PromethION. Quick spoke about how they are studying fecal microbiome transplants for ulcerative colitis and the evolution of the respiratory microbiome of cystic fibrosis. The team took very high molecular weight DNA using the Sambrook method and used the rapid transposase library kit. Quick’s method is to saturate the transposase with DNA to get the longest reads. Quick’s example with an E. coli strain produced an N50 of 63 kb, and they assembled the genome in eight reads! Quick said that “these ultra-long reads can really trivialize assembly.” However, microbiome samples are complex. Ligation libraries suffered some fragmentation during the bead cleanups. The Long Read Club has developed a bead-free ligation method. Quick spoke about using the Zymo microbial community standard to develop methods and improve them. Quick noted that the standards sometimes come in DNA/RNA Safe reagent that does cause some lysis. Quick’s group generated data from the Zymo standard community and the log version on the GridION and PromethION. Using the log community, they were able to determine abundance and accuracy. With the PromethION data, there is enough coverage to assemble. Some organisms were only detected. This was all done with the bead-beating approach. Quick also split lysate into two purifications: spin column and magnetic beads. Longer fragments were obtained with the magnetic bead system. The MetaPolyzyme was used to generate spheroplasts with a two-hour incubation. The three peaks challenge is to start with the sample in DNA/RNA Shield, perform chemical and enzymatic lysis, treat with MetaPolyzyme, and then bead beating. The supernatants are removed at each step and then pooled for final purification. Interestingly, the yeast in the community required bead beating. Quick encouraged using MetaPolyzyme to improve microbial communities’ read length. Quick shared data with R10 flow cells. We should use MetaPolyzyme and the Zymo HMW kit for some soil and fecal samples.
