A study is being conducted to test DArTseq genotyping technology for use in bacterial identification and strain typing. This technology involves sequencing of complexity reduced genomes derived by digestion with pairs of restriction enzymes and subsequent PCR amplification. The amplified restriction fragments are then sequenced. The aim of this study is to test different pairs of restriction enzymes to determine the number of sequenced fragments generated from the different combinations.
A total of 33 previously identified bacterial isolates provided by the Department of Microbiology in the Canberra Hospital were genotyped using DArTseq. Restriction endonuclease pairs used were: PstI – HpaII, PstI – MseI and MseI – HpaII. Samples were sequenced to give approximately 80,000 short reads per sample and fragment lengths between 40 to 69 bp. Sequences obtained were used for BLAST against the NCBI nt database collection. A set of bioinformatics tools were developed to process data and obtain the identity of the closest matching strain from amongst the NCBI nt database. Once the closest matching fully sequenced genome was identified, BLAST was performed against the identified genome.
Sequencing results from 33 samples combined, produced the following number of fragments for PstI-HpaII, PstI-MseI and MseI-HpaII: 5860, 5271 and 14746 tags respectively. The uniquely aligned restriction fragments for Enterococcus faecium yielded 374, 660 and 1888 fragments respectively; similarly for Staphylococcus aureus yields were 108, 527 and 1781 fragments respectively.
Methods gave a different number of sequenced tags for BLAST alignment, even at the same total sequencing depth; however, they resulted in the same identification results at species level and sometimes differed at the strain level. The method which yielded the largest number of fragments may potentially offer finer resolution for identification purposes as well as further information which may be applicable to strain typing due to the higher genome coverage obtained.
A study is being conducted to test DArTseq genotyping technology for use in bacterial identification and strain typing. This technology involves sequencing of complexity reduced genomes derived by digestion with pairs of restriction enzymes and subsequent PCR amplification. The amplified restriction fragments are then sequenced. The aim of this study is to test different pairs of restriction enzymes to determine the number of sequenced fragments generated from the different combinations.
A total of 33 previously identified bacterial isolates provided by the Department of Microbiology in the Canberra Hospital were genotyped using DArTseq. Restriction endonuclease pairs used were: PstI – HpaII, PstI – MseI and MseI – HpaII. Samples were sequenced to give approximately 80,000 short reads per sample and fragment lengths between 40 to 69 bp. Sequences obtained were used for BLAST against the NCBI nt database collection. A set of bioinformatics tools were developed to process data and obtain the identity of the closest matching strain from amongst the NCBI nt database. Once the closest matching fully sequenced genome was identified, BLAST was performed against the identified genome.
Sequencing results from 33 samples combined, produced the following number of fragments for PstI-HpaII, PstI-MseI and MseI-HpaII: 5860, 5271 and 14746 tags respectively. The uniquely aligned restriction fragments for Enterococcus faecium yielded 374, 660 and 1888 fragments respectively; similarly ...
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