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A new platform for ultra-high density Staphylococcus aureus transposon libraries.

Santiago M, Matano LM, Moussa SH, Gilmore MS, Walker S, Meredith TC - BMC Genomics (2015)

Bottom Line: Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress.We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures.The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. marinasantiago@fas.harvard.edu.

ABSTRACT

Background: Staphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology. High throughput, parallel-sequencing methods for analyzing transposon mutant libraries have the potential to revolutionize studies of S. aureus, but the genetic tools to take advantage of the power of next generation sequencing have not been fully developed.

Results: Here we report a phage-based transposition system to make ultra-high density transposon libraries for genome-wide analysis of mutant fitness in any Φ11-transducible S. aureus strain. The high efficiency of the delivery system has made it possible to multiplex transposon cassettes containing different regulatory elements in order to make libraries in which genes are over- or under-expressed as well as deleted. By incorporating transposon-specific barcodes into the cassettes, we can evaluate how mutations and changes in gene expression levels affect fitness in a single sequencing data set. Demonstrating the power of the system, we have prepared a library containing more than 690,000 unique insertions. Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress. We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures.

Conclusions: The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

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Related in: MedlinePlus

Protocol for the preparation of a high quality transposon DNA library for NGS. (A) (1) Genomic DNA is isolated and digested with NotI. High molecular weight DNA is selectively precipitated using an 8%PEG + NaCl solution, and transposon-plasmid junctions (106 bp) are removed in the supernatant. A biotinylated dsDNA adapter with NotI overhang is ligated (2) before digestion with MmeI, which cuts non-specifically 20 bp from its recognition site within ITR2 into the genome to liberate biotinylated-transposon-genome junctions as short DNA fragments (114 bp). (3). Biotinylated fragments are bound to streptavidin beads (4), and an Illumina sequencing primer adapter containing an indexing barcode and MmeI compatible ends is ligated (5). Primers annealing to the P7 site and the Illumina sequencing primer adapter sequence (with a P5 site overhang) are used to PCR amplify the transposon-genome junctions (6), agarose gel purified, and submitted for Illumina sequencing (7). NGS reads capture both the 16-bp of flanking genomic DNA as well as the transposon donor specific barcode located between the P7 and ITR2. (B) Fragments arising from transposon-plasmid junctions are removed by size selective PEG-NaCl precipitation, while the remaining fragments lack both P7 annealing sites and MmeI sites for ligation of the Illumina sequencing primer adapter. These fragments are therefore not amplified in step (6) of 4A. (C) By performing the size-selective precipitation on a 1 kb DNA ladder, we show that small 300 bp fragments of DNA are retained in the solution (SN), while larger DNA is precipitated (P). (D) Six transposon donor constructs were multiplexed and designed to attenuate expression of genes proximal to the insertion site according to the regulatory elements located at the ends of the transposon backbone. Each donor can be identified from NGS reads by the unique 3 bp barcode.
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Fig4: Protocol for the preparation of a high quality transposon DNA library for NGS. (A) (1) Genomic DNA is isolated and digested with NotI. High molecular weight DNA is selectively precipitated using an 8%PEG + NaCl solution, and transposon-plasmid junctions (106 bp) are removed in the supernatant. A biotinylated dsDNA adapter with NotI overhang is ligated (2) before digestion with MmeI, which cuts non-specifically 20 bp from its recognition site within ITR2 into the genome to liberate biotinylated-transposon-genome junctions as short DNA fragments (114 bp). (3). Biotinylated fragments are bound to streptavidin beads (4), and an Illumina sequencing primer adapter containing an indexing barcode and MmeI compatible ends is ligated (5). Primers annealing to the P7 site and the Illumina sequencing primer adapter sequence (with a P5 site overhang) are used to PCR amplify the transposon-genome junctions (6), agarose gel purified, and submitted for Illumina sequencing (7). NGS reads capture both the 16-bp of flanking genomic DNA as well as the transposon donor specific barcode located between the P7 and ITR2. (B) Fragments arising from transposon-plasmid junctions are removed by size selective PEG-NaCl precipitation, while the remaining fragments lack both P7 annealing sites and MmeI sites for ligation of the Illumina sequencing primer adapter. These fragments are therefore not amplified in step (6) of 4A. (C) By performing the size-selective precipitation on a 1 kb DNA ladder, we show that small 300 bp fragments of DNA are retained in the solution (SN), while larger DNA is precipitated (P). (D) Six transposon donor constructs were multiplexed and designed to attenuate expression of genes proximal to the insertion site according to the regulatory elements located at the ends of the transposon backbone. Each donor can be identified from NGS reads by the unique 3 bp barcode.

Mentions: Our optimized sample preparation procedure for transposon mapping is outlined in Figure 4A, and a detailed version can be found in the Additional file 1. Briefly, genomic DNA is extracted from a pooled transposon library and then digested with NotI followed by a size-selective poly-ethylene glycol (PEG) precipitation to remove liberated plasmid-transposon junctions (Figure 4B) [40,41]. A PCR-based quality control check is performed to confirm that background due to transposon-plasmid junctions is minimal (Additional file 1: Figure S3A). A biotinylated dsDNA adapter containing a NotI compatible overhang is then ligated and the DNA is digested with MmeI. The transposon-genome junctions with 2-base overhangs are bound to streptavidin dynabeads and ligated to an adapter containing the indexing barcode and priming site for the Illumina sequencing primer. Primers containing the P5 and P7 sites are then used to amplify the transposon-genome junctions bound to the streptavidin dynabeads. The fragments are run on a 2% agarose gel to confirm size, gel extracted, and multiplexed with other samples prior to sequencing. Using this protocol, we routinely reduced the amount of contaminating plasmid-transposon reads to less than 1%. To confirm the quality of the DNA insert library for NGS, we developed a quality control procedure for determining level of background due to transposon-plasmid junctions (Additional file 1: Figure S3B). This strategy, which utilizes restriction enzyme digestion followed by size-selective precipitations, can be generalized to other bacterial species and transposon library sequencing strategies to prepare transposon libraries for NGS.Figure 4


A new platform for ultra-high density Staphylococcus aureus transposon libraries.

Santiago M, Matano LM, Moussa SH, Gilmore MS, Walker S, Meredith TC - BMC Genomics (2015)

Protocol for the preparation of a high quality transposon DNA library for NGS. (A) (1) Genomic DNA is isolated and digested with NotI. High molecular weight DNA is selectively precipitated using an 8%PEG + NaCl solution, and transposon-plasmid junctions (106 bp) are removed in the supernatant. A biotinylated dsDNA adapter with NotI overhang is ligated (2) before digestion with MmeI, which cuts non-specifically 20 bp from its recognition site within ITR2 into the genome to liberate biotinylated-transposon-genome junctions as short DNA fragments (114 bp). (3). Biotinylated fragments are bound to streptavidin beads (4), and an Illumina sequencing primer adapter containing an indexing barcode and MmeI compatible ends is ligated (5). Primers annealing to the P7 site and the Illumina sequencing primer adapter sequence (with a P5 site overhang) are used to PCR amplify the transposon-genome junctions (6), agarose gel purified, and submitted for Illumina sequencing (7). NGS reads capture both the 16-bp of flanking genomic DNA as well as the transposon donor specific barcode located between the P7 and ITR2. (B) Fragments arising from transposon-plasmid junctions are removed by size selective PEG-NaCl precipitation, while the remaining fragments lack both P7 annealing sites and MmeI sites for ligation of the Illumina sequencing primer adapter. These fragments are therefore not amplified in step (6) of 4A. (C) By performing the size-selective precipitation on a 1 kb DNA ladder, we show that small 300 bp fragments of DNA are retained in the solution (SN), while larger DNA is precipitated (P). (D) Six transposon donor constructs were multiplexed and designed to attenuate expression of genes proximal to the insertion site according to the regulatory elements located at the ends of the transposon backbone. Each donor can be identified from NGS reads by the unique 3 bp barcode.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Protocol for the preparation of a high quality transposon DNA library for NGS. (A) (1) Genomic DNA is isolated and digested with NotI. High molecular weight DNA is selectively precipitated using an 8%PEG + NaCl solution, and transposon-plasmid junctions (106 bp) are removed in the supernatant. A biotinylated dsDNA adapter with NotI overhang is ligated (2) before digestion with MmeI, which cuts non-specifically 20 bp from its recognition site within ITR2 into the genome to liberate biotinylated-transposon-genome junctions as short DNA fragments (114 bp). (3). Biotinylated fragments are bound to streptavidin beads (4), and an Illumina sequencing primer adapter containing an indexing barcode and MmeI compatible ends is ligated (5). Primers annealing to the P7 site and the Illumina sequencing primer adapter sequence (with a P5 site overhang) are used to PCR amplify the transposon-genome junctions (6), agarose gel purified, and submitted for Illumina sequencing (7). NGS reads capture both the 16-bp of flanking genomic DNA as well as the transposon donor specific barcode located between the P7 and ITR2. (B) Fragments arising from transposon-plasmid junctions are removed by size selective PEG-NaCl precipitation, while the remaining fragments lack both P7 annealing sites and MmeI sites for ligation of the Illumina sequencing primer adapter. These fragments are therefore not amplified in step (6) of 4A. (C) By performing the size-selective precipitation on a 1 kb DNA ladder, we show that small 300 bp fragments of DNA are retained in the solution (SN), while larger DNA is precipitated (P). (D) Six transposon donor constructs were multiplexed and designed to attenuate expression of genes proximal to the insertion site according to the regulatory elements located at the ends of the transposon backbone. Each donor can be identified from NGS reads by the unique 3 bp barcode.
Mentions: Our optimized sample preparation procedure for transposon mapping is outlined in Figure 4A, and a detailed version can be found in the Additional file 1. Briefly, genomic DNA is extracted from a pooled transposon library and then digested with NotI followed by a size-selective poly-ethylene glycol (PEG) precipitation to remove liberated plasmid-transposon junctions (Figure 4B) [40,41]. A PCR-based quality control check is performed to confirm that background due to transposon-plasmid junctions is minimal (Additional file 1: Figure S3A). A biotinylated dsDNA adapter containing a NotI compatible overhang is then ligated and the DNA is digested with MmeI. The transposon-genome junctions with 2-base overhangs are bound to streptavidin dynabeads and ligated to an adapter containing the indexing barcode and priming site for the Illumina sequencing primer. Primers containing the P5 and P7 sites are then used to amplify the transposon-genome junctions bound to the streptavidin dynabeads. The fragments are run on a 2% agarose gel to confirm size, gel extracted, and multiplexed with other samples prior to sequencing. Using this protocol, we routinely reduced the amount of contaminating plasmid-transposon reads to less than 1%. To confirm the quality of the DNA insert library for NGS, we developed a quality control procedure for determining level of background due to transposon-plasmid junctions (Additional file 1: Figure S3B). This strategy, which utilizes restriction enzyme digestion followed by size-selective precipitations, can be generalized to other bacterial species and transposon library sequencing strategies to prepare transposon libraries for NGS.Figure 4

Bottom Line: Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress.We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures.The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. marinasantiago@fas.harvard.edu.

ABSTRACT

Background: Staphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology. High throughput, parallel-sequencing methods for analyzing transposon mutant libraries have the potential to revolutionize studies of S. aureus, but the genetic tools to take advantage of the power of next generation sequencing have not been fully developed.

Results: Here we report a phage-based transposition system to make ultra-high density transposon libraries for genome-wide analysis of mutant fitness in any Φ11-transducible S. aureus strain. The high efficiency of the delivery system has made it possible to multiplex transposon cassettes containing different regulatory elements in order to make libraries in which genes are over- or under-expressed as well as deleted. By incorporating transposon-specific barcodes into the cassettes, we can evaluate how mutations and changes in gene expression levels affect fitness in a single sequencing data set. Demonstrating the power of the system, we have prepared a library containing more than 690,000 unique insertions. Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress. We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures.

Conclusions: The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

Show MeSH
Related in: MedlinePlus