Nadie Holmes from the University of Nottingham in the UK presented at London Calling 2019 on “Extracting megabase DNA.” They work with DeepSeq providing training and core sequencing support. Holmes worked on collaborative projects to optimize ultra-long sequencing. They shared data from a 1.3 megabase read. They use the protocol developed by Josh Quick to extract megabase DNA. With 5 x 107 cells, they obtain microgram amounts. However, these DNA preps are challenging to work with. Holmes uses the Agilent Tape Station Genomic Screen Tape to check DNA preparations. Sage Science Pippin Pulse provides a good resolution between 5-430 kb. Holmes uses 15 ug of DNA and the RAD004 Ultra long Library Prep Protocol that Quick and team developed. They start with 15 ug of DNA in 18 ul of water and include 2 ul of FRM. They then slowly mix 10X with a wide bore pipette tip and incubate for one minute at 30 and one minute at 80 C. They incubate at room temperature with RAP and then load the library with water instead of loading beads. At every step, they pipette gently 5-10X. They then wait 10 minutes for “tethering” after loading. However, they were getting low yields because of clogging. The output Holmes mentioned depended on the sample but between 1-2 Gbases. The N50s with this prep were above 100 kb! BulkVis was developed to resolve fragmented ultra-long reads. With this correction, they identified a 2.5 Mb read. Holmes and the team tried this approach on Flongle and PromethION flow cells. It worked with Flongle flow cells to obtain N50s above 100 kb. On PromethION flow cells, the throughput was low, likely due to the rapid blocking of pores. Holmes has sequenced several interesting samples, including a deceased koala’s DNA. Holmes spoke about cleaning samples after extraction to remove shorter fragments. Holmes did note that shearing DNA helps with sequencing throughput. They mentioned Circulomics and the older version of the Short Read Eliminator. Holmes shared tips I had not considered, such as not using the beads for loading.
