Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
BAC SUDOKU SEQUENCING STRATEGY FOR IN
SILICO SCREENING OF LARGE-INSERT SOIL
METAGENOMIC LIBRARIES
Wednesday, 1st June 18:30 La Fonda NM Room (1st floor) Poster (PS‐1a.19)
Scott Monsma 1 , Jinglie Zhou 2 , Alinne Pereira 2 , Blaine Pfeifer 3 , Timothy Bugni 4 , Scott R.
Santos 2 , Megan Niebauer 1 , Erin Ferguson 1 , Ronald Godiska 1 , Chengcang Wu 5 , David
Mead 1 , Mark R. Liles 2
1 Lucigen Corporation, 2 Auburn University, 3 State University of New York at Buffalo,
4 University of Wisconsin‐Madison, 5 Intact Genomics Inc
Soil microorganisms express diverse bioactive natural products; however, the majority of soil microbes
are recalcitrant to cultivation. We are using a metagenomic approach to bypass cultivation
and directly capture the DNA from diverse microbial genomes in natural environments such as
soils. A metagenomic library from an agricultural soil (Cullars Rotation, Auburn, AL) was constructed
in a broad host‐range BAC vector that contained 19,200 clones with an average insert size
of 110kb. Screening was accomplished using strategy termed BAC Sudoku sequencing, wherein a
pooling strategy is used to multiplex the results, while still providing the ID of individual clones.
BAC clones were sequenced in pools (row, column, and plate) using indexed primers and paired
end reads on an Illumina HiSeq. Contigs were assembled for each pool and screened for secondary
metabolite gene clusters using antiSMASH, resulting in identification of >1000 novel PKS/NRPS
pathway‐containing clones. The cloned pathways are very divergent from known pathways, with the
GC content varying from 41 to 76% and the amino acid identity of the KS domains ranging from
32 to 83% to the best matching BLAST hit. Expression of these PKS pathway‐containing clones
in E. coli strain BTRA (engineered for polyketide precursor expression) has resulted in multiple
clones with evidence for heterologous expression of a cloned PKS pathway. These results indicate
a high degree of unique sequence space has been recovered from large‐insert metagenomic clones
and a subset of these clones are capable of being heterologously expressed to produce secondary
metabolites, thereby expanding our available resources for natural product discovery.
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