Human immunodeficiency virus (HIV) is a rapidly evolving pathogen that causes chronic infections, ... more Human immunodeficiency virus (HIV) is a rapidly evolving pathogen that causes chronic infections, so genetic diversity within a single infection can be very high. High-throughput "deep" sequencing can now measure this diversity in unprecedented detail, particularly since it can be performed at different timepoints during an infection, and this offers a potentially powerful way to infer the evolutionary dynamics of the intra-host viral population. However, population genomic inference from HIV sequence data is challenging because of high rates of mutation and recombination, rapid demographic changes, and ongoing selective pressures. In this paper we develop a new method for inference using HIV deep sequencing data using an approach based on importance sampling of ancestral recombination graphs under a multi-locus coalescent model. The approach further extends recent progress in the approximation of so-called conditional sampling distributions, a quantity of key interest when a...
This month's Genome Watch examines how the increased availability of mammalian genomes pr... more This month's Genome Watch examines how the increased availability of mammalian genomes provides new insights into the interactions of endogenous retroviruses with other viruses and various hosts.
Philosophical transactions of the Royal Society of London. Series B, Biological sciences, Jan 5, 2015
Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune s... more Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune system dynamics. Much research in this nascent field has focused on resolving immune responses to viral infection. However, the dynamics of B-cell diversity in early HIV infection, and in response to anti-retroviral therapy, are still poorly understood. Here, we investigate these dynamics through bulk Ig sequencing of samples collected over 2 years from a group of eight HIV-1 infected patients, five of whom received anti-retroviral therapy during the first half of the study period. We applied previously published methods for visualizing and quantifying B-cell sequence diversity, including the Gini index, and compared their efficacy to alternative measures. While we found significantly greater clonal structure in HIV-infected patients versus healthy controls, within HIV patients, we observed no significant relationships between statistics of B-cell clonal expansion and clinical variables s...
The precise immune responses mediated by HLA class I molecules such as HLA-B*27:05 and HLA-B*57:0... more The precise immune responses mediated by HLA class I molecules such as HLA-B*27:05 and HLA-B*57:01 that protect against HIV disease progression remain unclear. We studied a CRF01_AE clade HIV infected donor-recipient transmission pair in which the recipient expressed both HLA-B*27:05 and HLA-B*57:01. Within 4.5 years of diagnosis, the recipient had progressed to meet criteria for antiretroviral therapy initiation. We employed ultra-deep sequencing of the full-length virus genome in both donor and recipient as an unbiased approach by which to identify specific viral mutations selected in association with progression. Using a heat map method to highlight differences in the viral sequences between donor and recipient, we demonstrated that the majority of the recipient's mutations outside of Env were within epitopes restricted by HLA-B*27:05 and HLA-B*57:01, including the well-studied Gag epitopes. The donor, who also expressed HLA alleles associated with disease protection, HLA-A*3...
The World Health Organization (WHO) International Standard for HIV-1 RNA nucleic acid assays was ... more The World Health Organization (WHO) International Standard for HIV-1 RNA nucleic acid assays was characterized by complete genome deep sequencing analysis. The entire coding sequence and flanking long terminal repeats (LTRs), including minority species, were assigned subtype B. This information will aid the design, development, and evaluation of HIV-1 RNA amplification assays.
An accurate genome assembly from short read sequencing data is critical for downstream analysis, ... more An accurate genome assembly from short read sequencing data is critical for downstream analysis, for example allowing investigation of variants within a sequenced population. However, assembling sequencing data from virus samples, especially RNA viruses, into a genome sequence is challenging due to the combination of viral population diversity and extremely uneven read depth caused by amplification bias in the inevitable reverse transcription and PCR amplification process of current methods. We developed IVA (Iterative Virus Assembler), a de novo assembler designed specifically for read pairs sequenced at highly variable depth from RNA virus samples. We tested IVA on datasets from 140 sequenced samples from HIV-1 or Influenza virus infected people and demonstrated that IVA outperforms all other virus de novo assemblers. Availability: The software runs under Linux, has the GPLv3 licence and is freely available from http://sanger-pathogens.github.io/iva CONTACT: iva@sanger.ac.uk.
Rabbit haemorrhagic disease (RHD) is usually a fatal disease in rabbits which has spread rapidly ... more Rabbit haemorrhagic disease (RHD) is usually a fatal disease in rabbits which has spread rapidly across the continents. While previous studies suggested persistence in rabbits to be an important factor in the epidemiology, the relevance of field virus infection of immune rabbits has not been investigated in experimentally infected animals before. This report describes for the first time the persistence of rabbit haemorrhagic disease virus (RHDV) genome for at least 15 weeks in rabbits immunized with an inactivated vaccine as well as a subunit vaccine and subsequently challenged with virulent RHDV. The viral RNA loads were determined by real-time reverse transcription-polymerase chain reaction. No conspicuous association of the detectable amount of RHDV RNA with the type of vaccine, the time after infection and--with one exception--the level of RHDV-specific antibodies in the immunized animals was observed. The results presented in this study are an urgent evidence for the existence of carrier animals as an important factor in the epidemiology of RHD.
An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards fo... more An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.
This month's Genome Watch highlights how deep sequencing was used to generate the... more This month's Genome Watch highlights how deep sequencing was used to generate the first full genomes of herpesviruses associated with a fatal disease in elephants.
Influenza A surface proteins H (haemagglutinin) and N (neuraminidase) occur in sixteen and nine d... more Influenza A surface proteins H (haemagglutinin) and N (neuraminidase) occur in sixteen and nine distinct genotypes, respectively. The need for a timely production of vaccinations in case of pandemics or seasonal epidemics requires rapid typing methods for the determination of these alleles. The aim of the present study was to develop and improve a rapid and economic assay for determining H and N subtypes of influenza A from patient samples. The assay is based on the hybridisation of labelled amplicons from H and N reverse transcriptase-PCRs using consensus primer pairs to subtype-specific probes on microtiterstripe-mounted DNA-microarrays. An algorithm for semi-automatic data interpretation of raw data and assignment to H and N subtypes was proposed. Altogether, 191 samples were genotyped. This included 134 patient and 44 reference samples as well as controls. Under routine conditions sensitivity and specificity proved to be comparable to conventional nested or real-time PCRs. At least 130 out of 147 array-positive samples were unambiguously assignable. This included all sixteen variants of H as well as all nine variants of N. Furthermore, eighty-two samples from the 2009/2010 "novel H1N1/swine flu" (SF)-outbreak were correctly identified.
Human immunodeficiency virus (HIV) is a rapidly evolving pathogen that causes chronic infections, ... more Human immunodeficiency virus (HIV) is a rapidly evolving pathogen that causes chronic infections, so genetic diversity within a single infection can be very high. High-throughput "deep" sequencing can now measure this diversity in unprecedented detail, particularly since it can be performed at different timepoints during an infection, and this offers a potentially powerful way to infer the evolutionary dynamics of the intra-host viral population. However, population genomic inference from HIV sequence data is challenging because of high rates of mutation and recombination, rapid demographic changes, and ongoing selective pressures. In this paper we develop a new method for inference using HIV deep sequencing data using an approach based on importance sampling of ancestral recombination graphs under a multi-locus coalescent model. The approach further extends recent progress in the approximation of so-called conditional sampling distributions, a quantity of key interest when a...
This month's Genome Watch examines how the increased availability of mammalian genomes pr... more This month's Genome Watch examines how the increased availability of mammalian genomes provides new insights into the interactions of endogenous retroviruses with other viruses and various hosts.
Philosophical transactions of the Royal Society of London. Series B, Biological sciences, Jan 5, 2015
Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune s... more Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune system dynamics. Much research in this nascent field has focused on resolving immune responses to viral infection. However, the dynamics of B-cell diversity in early HIV infection, and in response to anti-retroviral therapy, are still poorly understood. Here, we investigate these dynamics through bulk Ig sequencing of samples collected over 2 years from a group of eight HIV-1 infected patients, five of whom received anti-retroviral therapy during the first half of the study period. We applied previously published methods for visualizing and quantifying B-cell sequence diversity, including the Gini index, and compared their efficacy to alternative measures. While we found significantly greater clonal structure in HIV-infected patients versus healthy controls, within HIV patients, we observed no significant relationships between statistics of B-cell clonal expansion and clinical variables s...
The precise immune responses mediated by HLA class I molecules such as HLA-B*27:05 and HLA-B*57:0... more The precise immune responses mediated by HLA class I molecules such as HLA-B*27:05 and HLA-B*57:01 that protect against HIV disease progression remain unclear. We studied a CRF01_AE clade HIV infected donor-recipient transmission pair in which the recipient expressed both HLA-B*27:05 and HLA-B*57:01. Within 4.5 years of diagnosis, the recipient had progressed to meet criteria for antiretroviral therapy initiation. We employed ultra-deep sequencing of the full-length virus genome in both donor and recipient as an unbiased approach by which to identify specific viral mutations selected in association with progression. Using a heat map method to highlight differences in the viral sequences between donor and recipient, we demonstrated that the majority of the recipient's mutations outside of Env were within epitopes restricted by HLA-B*27:05 and HLA-B*57:01, including the well-studied Gag epitopes. The donor, who also expressed HLA alleles associated with disease protection, HLA-A*3...
The World Health Organization (WHO) International Standard for HIV-1 RNA nucleic acid assays was ... more The World Health Organization (WHO) International Standard for HIV-1 RNA nucleic acid assays was characterized by complete genome deep sequencing analysis. The entire coding sequence and flanking long terminal repeats (LTRs), including minority species, were assigned subtype B. This information will aid the design, development, and evaluation of HIV-1 RNA amplification assays.
An accurate genome assembly from short read sequencing data is critical for downstream analysis, ... more An accurate genome assembly from short read sequencing data is critical for downstream analysis, for example allowing investigation of variants within a sequenced population. However, assembling sequencing data from virus samples, especially RNA viruses, into a genome sequence is challenging due to the combination of viral population diversity and extremely uneven read depth caused by amplification bias in the inevitable reverse transcription and PCR amplification process of current methods. We developed IVA (Iterative Virus Assembler), a de novo assembler designed specifically for read pairs sequenced at highly variable depth from RNA virus samples. We tested IVA on datasets from 140 sequenced samples from HIV-1 or Influenza virus infected people and demonstrated that IVA outperforms all other virus de novo assemblers. Availability: The software runs under Linux, has the GPLv3 licence and is freely available from http://sanger-pathogens.github.io/iva CONTACT: iva@sanger.ac.uk.
Rabbit haemorrhagic disease (RHD) is usually a fatal disease in rabbits which has spread rapidly ... more Rabbit haemorrhagic disease (RHD) is usually a fatal disease in rabbits which has spread rapidly across the continents. While previous studies suggested persistence in rabbits to be an important factor in the epidemiology, the relevance of field virus infection of immune rabbits has not been investigated in experimentally infected animals before. This report describes for the first time the persistence of rabbit haemorrhagic disease virus (RHDV) genome for at least 15 weeks in rabbits immunized with an inactivated vaccine as well as a subunit vaccine and subsequently challenged with virulent RHDV. The viral RNA loads were determined by real-time reverse transcription-polymerase chain reaction. No conspicuous association of the detectable amount of RHDV RNA with the type of vaccine, the time after infection and--with one exception--the level of RHDV-specific antibodies in the immunized animals was observed. The results presented in this study are an urgent evidence for the existence of carrier animals as an important factor in the epidemiology of RHD.
An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards fo... more An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.
This month's Genome Watch highlights how deep sequencing was used to generate the... more This month's Genome Watch highlights how deep sequencing was used to generate the first full genomes of herpesviruses associated with a fatal disease in elephants.
Influenza A surface proteins H (haemagglutinin) and N (neuraminidase) occur in sixteen and nine d... more Influenza A surface proteins H (haemagglutinin) and N (neuraminidase) occur in sixteen and nine distinct genotypes, respectively. The need for a timely production of vaccinations in case of pandemics or seasonal epidemics requires rapid typing methods for the determination of these alleles. The aim of the present study was to develop and improve a rapid and economic assay for determining H and N subtypes of influenza A from patient samples. The assay is based on the hybridisation of labelled amplicons from H and N reverse transcriptase-PCRs using consensus primer pairs to subtype-specific probes on microtiterstripe-mounted DNA-microarrays. An algorithm for semi-automatic data interpretation of raw data and assignment to H and N subtypes was proposed. Altogether, 191 samples were genotyped. This included 134 patient and 44 reference samples as well as controls. Under routine conditions sensitivity and specificity proved to be comparable to conventional nested or real-time PCRs. At least 130 out of 147 array-positive samples were unambiguously assignable. This included all sixteen variants of H as well as all nine variants of N. Furthermore, eighty-two samples from the 2009/2010 "novel H1N1/swine flu" (SF)-outbreak were correctly identified.
Uploads
Papers by Astrid Gall