Duah Alkam, a Ph.D. student in Mark Smeltzer‘s and Dave Ussery‘s laboratories was invited to present her work at the Nanopore Community Meeting in New York. A description of her presentation from Oxford Nanopore Technologies is below.
Duah Alkam – PCR-free transposon sequencing (TnSeq): Cas9/dCas9-mediated transposon enrichment
Duah Alkam (University of Arkansas for Medical Sciences) opened the Targeted Sequencing breakout session by introducing the goal of her team: to find therapeutic targets for the prominent bacterial pathogen Staphylococcus aureus. Staphylococcus bacteria are a leading cause of healthcare-associated infections, with Methicillin-resistant S. aureus (MRSA) causing over 80,000 severe infections and over 11,000 deaths per year.
Duah and the team take a whole-genome screening approach to the search for novel therapeutics via transposon sequencing (TnSeq), enabling analysis of the genes that affect the fitness of microorganisms in a particular condition. She described how, in a transposon library, each cell is mutagenized by a transposon – a DNA segment that inserts into a gene, resulting in dysfunction. The transposon library used by Duah and her colleagues is in a background of a clinically-relevant strain of S. aureus; Duah gave an overview of a typical workflow, in which the libraries are grown in different conditions, then enriched for the region of interest. The current protocol for TnSeq requires PCR amplification of the transposon-library junctions prior to sequencing; Duah noted that this method has been used in many papers. However, Duah explained, there’s a problem with this method: inherent bias is introduced through the PCR steps. Looking to overcome this, Duah asked: “can we enrich for the transposon via a PCR-free method?”. Here, Duah explained, the team turned to Oxford Nanopore and decided to explore PCR-free dCas9/Cas9-mediated transposon enrichment.
Duah outlined the four methods tested to investigate Cas9-mediated enrichment and nanopore sequencing of S. aureus TnSeq libraries; in each case, samples were prepared for sequencing without PCR using the Ligation Sequencing Kit and sequenced on the MinION device.
In the first condition, the TnSeq library was sequenced without any enrichment. This no-enrichment condition resulted in just 8,566, or 0.2% of the 5.1m reads representing transposons, as expected – transposons are “pretty rare in the library”. This confirmed the need for enrichment of the transposons.
In the second condition, CRISPR/Cas9 was used to induce a double-stranded cut targeting transposon insertion sites, then the library was sequenced. Duah outlined the CRISPR/Cas9-mediated enrichment method: firstly, the 5′ ends of all strands in the sample are dephosphorylated. Next, the Cas9 enzyme is directed to and cleaves DNA at transposon sites via RNAs including custom transposon-specific probes. This reveals phosphorylated ends, to which sequencing adapters can be ligated so that the enriched DNA can be sequenced. In this dataset, transposons were successfully enriched, representing 45% of reads in sequencing – “we were very excited when we saw this” – but sequencing yield was depleted, so that this was comprised of 3.7k reads.
To investigate whether yield could be improved, in the third condition, dCas9 – “dead” Cas9, an inactive form of the enzyme which does not cut the DNA – and biotinylated RNA probes were used, so that the biotinylated targets could be captured using Streptavidin beads then prepared for sequencing. Whilst this protocol resulted in only 0.8% transposon reads, sequencing output was higher, resulting in 94.6k reads.
Aiming to make the most of the enrichment from Cas9 and yield from dCas9, in the last condition, a combination of these methods were used. dCas9 and biotinylated probes were first used to capture the DNA, then active Cas9 was used to cleave the transposons at a single site prior to sequencing. Here, enrichment was lower than when using Cas9 alone, with 31% of reads representing transposons, but as sequencing output was higher, 19.4k reads were on-target.
Summarising, Duah concluded that she and her team had successfully devised and utilized a PCR-free method of transposon enrichment from a TnSeq S. aureus library, representing the first time this has been achieved without PCR. Furthermore, the use of dCas9 in pulldown prior to cleavage by Cas9 improved sequencing yield, enabling a greater number of transposon reads. They are now optimizing their protocol to further improve sequencing yield and recover greater numbers of insertions.