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Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples.

Christiansen MT, Brown AC, Kundu S, Tutill HJ, Williams R, Brown JR, Holdstock J, Holland MJ, Stevenson S, Dave J, Tong CY, Einer-Jensen K, Depledge DP, Breuer J - BMC Infect. Dis. (2014)

Bottom Line: All samples were sequenced on the MiSeq platform.Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance.The sensitivity of the method was >10-fold higher than other reported methodologies.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection and Immunity University College London (UCL), London, WC1E 6BT, UK. m.christiansen@ucl.ac.uk.

ABSTRACT

Background: Chlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples.

Methods: C. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform.

Results: Full length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies.

Conclusions: The combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

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Related in: MedlinePlus

Coverage of reference genome and mean read depth in relation to totalC. trachomatisgenome copies input. The plot shows the amount of target DNA (C. trachomatis DNA) used in the library preparations and the percentage coverage of the reference genome obtained with the various target input. The bars (black, grey, and white) illustrate the minimum mean read depth obtained from each sample. The columns to the far left represent a sample which did not undergo whole-genome enrichment during library preparation (indicated with text - No enrichment).
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Fig4: Coverage of reference genome and mean read depth in relation to totalC. trachomatisgenome copies input. The plot shows the amount of target DNA (C. trachomatis DNA) used in the library preparations and the percentage coverage of the reference genome obtained with the various target input. The bars (black, grey, and white) illustrate the minimum mean read depth obtained from each sample. The columns to the far left represent a sample which did not undergo whole-genome enrichment during library preparation (indicated with text - No enrichment).

Mentions: The sensitivity of whole-genome enrichment of C. trachomatis DNA using SureSelectXT was evaluated using a series of dilutions of C. trachomatis genomic DNA extracted from a cultured sample. For each dilution human gDNA was used to bulk the sample to contain 3 μg of starting material prior to library preparation and sequencing. The proportion of sequence reads mapping to a C. trachomatis reference genome (F/SW4) was calculated for each dilution (Figure 3). The data showed a saturation of ~90% on-target reads irrespective of the amount of input C. trachomatis DNA. From approximately 48,000 C. trachomatis input genomes (based on qPCR of the genomic omcB gene) we obtained close to 100% coverage of the reference genome with a mean read depth of 20× and around 85% coverage of the reference genome at a mean read depth of 100× (Figure 4). With a ten-fold lower input (4,800 C. trachomatis genomes) we obtained 98% coverage of the reference genome but with a lower mean read depth (Figure 4). Using whole-genome enrichment, a high proportion of sequence reads mapping to C. trachomatis were obtained from all of the cultured samples and full length genomes were recovered, confirming that our 120-mer RNA oligonucleotide set was capable of enriching for strains from both biovars (Table 1).Figure 3


Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples.

Christiansen MT, Brown AC, Kundu S, Tutill HJ, Williams R, Brown JR, Holdstock J, Holland MJ, Stevenson S, Dave J, Tong CY, Einer-Jensen K, Depledge DP, Breuer J - BMC Infect. Dis. (2014)

Coverage of reference genome and mean read depth in relation to totalC. trachomatisgenome copies input. The plot shows the amount of target DNA (C. trachomatis DNA) used in the library preparations and the percentage coverage of the reference genome obtained with the various target input. The bars (black, grey, and white) illustrate the minimum mean read depth obtained from each sample. The columns to the far left represent a sample which did not undergo whole-genome enrichment during library preparation (indicated with text - No enrichment).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4233057&req=5

Fig4: Coverage of reference genome and mean read depth in relation to totalC. trachomatisgenome copies input. The plot shows the amount of target DNA (C. trachomatis DNA) used in the library preparations and the percentage coverage of the reference genome obtained with the various target input. The bars (black, grey, and white) illustrate the minimum mean read depth obtained from each sample. The columns to the far left represent a sample which did not undergo whole-genome enrichment during library preparation (indicated with text - No enrichment).
Mentions: The sensitivity of whole-genome enrichment of C. trachomatis DNA using SureSelectXT was evaluated using a series of dilutions of C. trachomatis genomic DNA extracted from a cultured sample. For each dilution human gDNA was used to bulk the sample to contain 3 μg of starting material prior to library preparation and sequencing. The proportion of sequence reads mapping to a C. trachomatis reference genome (F/SW4) was calculated for each dilution (Figure 3). The data showed a saturation of ~90% on-target reads irrespective of the amount of input C. trachomatis DNA. From approximately 48,000 C. trachomatis input genomes (based on qPCR of the genomic omcB gene) we obtained close to 100% coverage of the reference genome with a mean read depth of 20× and around 85% coverage of the reference genome at a mean read depth of 100× (Figure 4). With a ten-fold lower input (4,800 C. trachomatis genomes) we obtained 98% coverage of the reference genome but with a lower mean read depth (Figure 4). Using whole-genome enrichment, a high proportion of sequence reads mapping to C. trachomatis were obtained from all of the cultured samples and full length genomes were recovered, confirming that our 120-mer RNA oligonucleotide set was capable of enriching for strains from both biovars (Table 1).Figure 3

Bottom Line: All samples were sequenced on the MiSeq platform.Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance.The sensitivity of the method was >10-fold higher than other reported methodologies.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection and Immunity University College London (UCL), London, WC1E 6BT, UK. m.christiansen@ucl.ac.uk.

ABSTRACT

Background: Chlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples.

Methods: C. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform.

Results: Full length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies.

Conclusions: The combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

Show MeSH
Related in: MedlinePlus