Compositions and methods for identifying the essential genome of an organism

摘要

Compositions and methods are provided for the rapid and highly accurate identification of the entire essential genome of any organism under a given selection condition at a resolution of a few base pairs. An engineered transposon bearing an adapter sequence for ultra high throughput adaptor-based sequencing is employed for hyper-saturated transposon mutagenesis. Transposon junctions are subsequently isolated and collectively amplified through a shared parallel PCR strategy such that a second adaptor sequence is further incorporated into template DNA so that the first adaptor sequence and the second adaptor sequence flank the 5′ and 3′ regions of the sample DNA, respectively. Sample DNA is then sequenced in an ultra high-throughput adaptor-based DNA sequencer using adaptor primers. Transposon insertion sites are mapped onto the organism's genome, allowing for the algorithmic identification of essential genetic elements based on genomic transposition frequency.

主权项

1. A method for determining genetic elements of an organism's genome, the method comprising:
performing hyper-saturated transposon mutagenesis to generate an engineered transposon comprising a Tn5 O-end insertion primer having the sequence of SEQ ID NO: 3, and a Tn5 I-end primer having the sequence of SEQ ID NO. 2;
said hyper-saturated transposon mutagenesis having a transposition density from about 8 to 100 bp; selecting for viable mutants in the engineered transposon under a given selection condition; pooling the selected viable mutants; isolating transposon junctions through a shared parallel PCR strategy utilizing a first primer that is transposon specific, at least one semi-arbitrary primer, and at least one adaptor sequence primer, wherein the at least one semi-arbitrary primer is selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6; sequencing the isolated transposon junctions in the ultra high-throughput adaptor based DNA sequencer; mapping transposon insertion sites onto the organism's genome; and identifying the genetic elements based on frequency, locations and insertion orientation of the mapped transposon insertion sites.