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Accurate automated annotation of DNA sequences related to antibiotic resistance in Gram-Negative bacteria
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PATHOLOGY 2014 ABSTRACT SUPPLEMENT
implicated in a case of severe haemolytic disease of the fetus and newborn (HDFN). This study aimed to identify the genetic basis of SARAH. Methods: DNA samples were collected from nine family members (six SARAH positive) from three generations. Samples were exome sequenced using the Agilent SureSelect All Exon v4 þ UTR kit on an Illumina HiSeq2000. Sequence alignment was performed with Illumina CASAVA v1.8.2, mapping to the reference genome HG19. Results: Sequencing identified 73 782 SNVs common among all family members. SNV filtering excluded SNVs with a NCBI dbSNP ID, non-protein coding SNVs, and SNVs not present in all six positive family members only. Following filtering, one SNV in the Glycophorin A (GYPA) gene was identified. This SNV is not in the 1000 Genomes Project (1000G) or Exome Sequencing Project (ESP6500) databases, further confirming this SNV is novel and of low-frequency. Discussion: A genetic basis for SARAH was identified by utilising next generation sequencing of related individuals. This approach has application in investigations relating to other rare phenotypes or diseases of unknown genetic basis. ACCURATE AUTOMATED ANNOTATION OF DNA SEQUENCES RELATED TO ANTIBIOTIC RESISTANCE IN GRAM-NEGATIVE BACTERIA G. Tsafnat1 and S. Partridge2 1Centre for Health Informatics, University of New South Wales, and 2Centre for Infectious Diseases and Microbiology, Westmead Hospital, Sydney, NSW, Australia Gram-negative bacteria resistant to multiple antibiotics are an increasing global health problem. This resistance is largely due to resistance genes ‘captured’ by mobile genetic elements that move them between DNA molecules in the same cell. Multiresistance spreads because genes conferring resistance to different classes of antibiotics and associated mobile elements are found in complex modular clusters on resistance plasmids that can transfer between cells, including different species. Increasing numbers of such plasmids are being sequenced, including by next-generation methods, but are often poorly annotated as current analysis software only gives general names to genes and suggest putative functions. We have developed an automated annotation system (Attacca) for DNA sequences relating to antibiotic resistance in Gram-negative bacteria that accurately and precisely annotates resistance genes and associated mobile elements. Attacca uses a database of relevant ‘features’ and computational grammars derived known rules about the behaviour of different mobile elements to annotate fragments of features and find informative signals that give information about movement of mobile elements. Attacca also creates diagrams of annotated sequences that facilitate detailed comparative analysis to help understand the evolution and spread of multi-resistance. We plan to make Attacca available via a website. HYPERSPECTRAL IMAGING OF BRUISES Claire G. Ross1, Carley Chwal2, Jeffrey A. Beckstead2, Roger W. Byard3 and Neil E. I. Langlois3 1SA Pathology, Department of Anatomical Pathology, The Queen Elizabeth Hospital, Adelaide, SA, Australia, 2Chemimage Corporation, Pittsburgh, PA, USA, and 3Forensic Science SA and University of Adelaide, Adelaide, SA, Australia
Pathology (2014), 46(S1)
Introduction: Hyperspectral imaging combines a camera with a tunable optical filter to record reflectance spectrophotometry data over a range of wavelengths for each image point. The data can be mathematically processed. Bruises comprise extravasated blood (haemoglobin) in the skin that is broken down to haemosiderin and bilirubin, the presence of which can be demonstrated by analysis of hyperspectral data. Aims: To develop hyperspectral image data processing to demonstrate the spatial distribution of bruises and their temporal changes. Method: Bruise-type injuries were created by injection of blood into skin of rats (with Animal Ethics Approval1). Hyperspectral images were captured using a Chemimage HSI examiner 100 QD at a range of time points. Hyperspectral data were processed to analyse the first derivative to quantify the accumulation of degradation products of haemoglobin; tissue was also examined histologically. Results: Hyperspectral images were successfully obtained; however, initial processing did not provide a defined correlation with time or histological changes. Discussion: Initial results using simple analysis of first derivative values did not produce a clear correlation with accumulation of haemoglobin degradation products over time. Advanced processing techniques will be tested and the outcome of these will be presented. Reference 1. Ross C, Byard RW. Langlois NEI. Does the intensity of the inflammatory reaction in a bruise depend on its proximity to the site of trauma? Forensic Sci Med Pathol 2013; 9: 358–62.
QUADRUPLEX REAL-TIME PCR USED TO INVESTIGATE A NEW STAPHYLOCOCCUS AUREUS GENE HOMOLOGUE mecALGA251 (DESIGNATED mecC) IN HUMAN AND BOVINE POPULATION; IN ADDITION TO nuc, LukS-PV AND mecA A. Yanga1, T. Badrick1, K. Ashton1 and S. Byrne2 1Faculty of Health Science and Medicine, Bond University, Robina, and 2Department of Clinical Microbiology, Sullivan Nicolaides Pathology, Taringa, Brisbane, Qld, Australia Background: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of hospital-associated (HA-MRSA) and community-associated (CA-MRSA) infections. It is also seen in the livestock population (livestock-associated MRSA). The emergence of bovine and human MRSA isolates carrying a new (SCCmec) element (type XI) introduced a new challenge for clinical microbiologists. This new SCCmec element contains a novel mecA homologue designated mecC (formerly known as mecALGA251) which is undetectable by conventional molecular approaches. Aim: The aim of this study was to investigate the prevalence of mecC in Australia using a quadruplex real-time PCR described by Pichon et al.1 that detects nuc, mecA, lukS-PV and mecC gene. Results: The assay was validated using 70 samples and no interference was observed when samples were run in singleplex and multiplex with no significant change in the C(t) values. The introduction of a synthetic mecC shows no interference with the performance of the other primers. Following successful validation, the quadruplex real-time PCR assay will be used to screen for nuc, mecA, lukS-PV and mecC. The results will be discussed further as data are being collected.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
PATHOLOGY 2014 ABSTRACT SUPPLEMENT
implicated in a case of severe haemolytic disease of the fetus and newborn (HDFN). This study aimed to identify the genetic basis of SARAH. Methods: DNA samples were collected from nine family members (six SARAH positive) from three generations. Samples were exome sequenced using the Agilent SureSelect All Exon v4 þ UTR kit on an Illumina HiSeq2000. Sequence alignment was performed with Illumina CASAVA v1.8.2, mapping to the reference genome HG19. Results: Sequencing identified 73 782 SNVs common among all family members. SNV filtering excluded SNVs with a NCBI dbSNP ID, non-protein coding SNVs, and SNVs not present in all six positive family members only. Following filtering, one SNV in the Glycophorin A (GYPA) gene was identified. This SNV is not in the 1000 Genomes Project (1000G) or Exome Sequencing Project (ESP6500) databases, further confirming this SNV is novel and of low-frequency. Discussion: A genetic basis for SARAH was identified by utilising next generation sequencing of related individuals. This approach has application in investigations relating to other rare phenotypes or diseases of unknown genetic basis. ACCURATE AUTOMATED ANNOTATION OF DNA SEQUENCES RELATED TO ANTIBIOTIC RESISTANCE IN GRAM-NEGATIVE BACTERIA G. Tsafnat1 and S. Partridge2 1Centre for Health Informatics, University of New South Wales, and 2Centre for Infectious Diseases and Microbiology, Westmead Hospital, Sydney, NSW, Australia Gram-negative bacteria resistant to multiple antibiotics are an increasing global health problem. This resistance is largely due to resistance genes ‘captured’ by mobile genetic elements that move them between DNA molecules in the same cell. Multiresistance spreads because genes conferring resistance to different classes of antibiotics and associated mobile elements are found in complex modular clusters on resistance plasmids that can transfer between cells, including different species. Increasing numbers of such plasmids are being sequenced, including by next-generation methods, but are often poorly annotated as current analysis software only gives general names to genes and suggest putative functions. We have developed an automated annotation system (Attacca) for DNA sequences relating to antibiotic resistance in Gram-negative bacteria that accurately and precisely annotates resistance genes and associated mobile elements. Attacca uses a database of relevant ‘features’ and computational grammars derived known rules about the behaviour of different mobile elements to annotate fragments of features and find informative signals that give information about movement of mobile elements. Attacca also creates diagrams of annotated sequences that facilitate detailed comparative analysis to help understand the evolution and spread of multi-resistance. We plan to make Attacca available via a website. HYPERSPECTRAL IMAGING OF BRUISES Claire G. Ross1, Carley Chwal2, Jeffrey A. Beckstead2, Roger W. Byard3 and Neil E. I. Langlois3 1SA Pathology, Department of Anatomical Pathology, The Queen Elizabeth Hospital, Adelaide, SA, Australia, 2Chemimage Corporation, Pittsburgh, PA, USA, and 3Forensic Science SA and University of Adelaide, Adelaide, SA, Australia
Pathology (2014), 46(S1)
Introduction: Hyperspectral imaging combines a camera with a tunable optical filter to record reflectance spectrophotometry data over a range of wavelengths for each image point. The data can be mathematically processed. Bruises comprise extravasated blood (haemoglobin) in the skin that is broken down to haemosiderin and bilirubin, the presence of which can be demonstrated by analysis of hyperspectral data. Aims: To develop hyperspectral image data processing to demonstrate the spatial distribution of bruises and their temporal changes. Method: Bruise-type injuries were created by injection of blood into skin of rats (with Animal Ethics Approval1). Hyperspectral images were captured using a Chemimage HSI examiner 100 QD at a range of time points. Hyperspectral data were processed to analyse the first derivative to quantify the accumulation of degradation products of haemoglobin; tissue was also examined histologically. Results: Hyperspectral images were successfully obtained; however, initial processing did not provide a defined correlation with time or histological changes. Discussion: Initial results using simple analysis of first derivative values did not produce a clear correlation with accumulation of haemoglobin degradation products over time. Advanced processing techniques will be tested and the outcome of these will be presented. Reference 1. Ross C, Byard RW. Langlois NEI. Does the intensity of the inflammatory reaction in a bruise depend on its proximity to the site of trauma? Forensic Sci Med Pathol 2013; 9: 358–62.
QUADRUPLEX REAL-TIME PCR USED TO INVESTIGATE A NEW STAPHYLOCOCCUS AUREUS GENE HOMOLOGUE mecALGA251 (DESIGNATED mecC) IN HUMAN AND BOVINE POPULATION; IN ADDITION TO nuc, LukS-PV AND mecA A. Yanga1, T. Badrick1, K. Ashton1 and S. Byrne2 1Faculty of Health Science and Medicine, Bond University, Robina, and 2Department of Clinical Microbiology, Sullivan Nicolaides Pathology, Taringa, Brisbane, Qld, Australia Background: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of hospital-associated (HA-MRSA) and community-associated (CA-MRSA) infections. It is also seen in the livestock population (livestock-associated MRSA). The emergence of bovine and human MRSA isolates carrying a new (SCCmec) element (type XI) introduced a new challenge for clinical microbiologists. This new SCCmec element contains a novel mecA homologue designated mecC (formerly known as mecALGA251) which is undetectable by conventional molecular approaches. Aim: The aim of this study was to investigate the prevalence of mecC in Australia using a quadruplex real-time PCR described by Pichon et al.1 that detects nuc, mecA, lukS-PV and mecC gene. Results: The assay was validated using 70 samples and no interference was observed when samples were run in singleplex and multiplex with no significant change in the C(t) values. The introduction of a synthetic mecC shows no interference with the performance of the other primers. Following successful validation, the quadruplex real-time PCR assay will be used to screen for nuc, mecA, lukS-PV and mecC. The results will be discussed further as data are being collected.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.