Limits...
A comprehensive next generation sequencing-based virome assessment in brain tissue suggests no major virus - tumor association.

Strong MJ, Blanchard E, Lin Z, Morris CA, Baddoo M, Taylor CM, Ware ML, Flemington EK - Acta Neuropathol Commun (2016)

Bottom Line: For example, all of the sporadic low level HCMV reads were found to map to the immediate early promoter intimating that they likely originated from laboratory expression vector contamination.This analysis argues against associations between most known viruses and GBM or mengiomas.Nevertheless, there may be a low percentage association between HPV and/or hepatitis B and LGGs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Tulane University, New Orleans, LA, 70112, USA.

ABSTRACT
Next generation sequencing (NGS) can globally interrogate the genetic composition of biological samples in an unbiased yet sensitive manner. The objective of this study was to utilize the capabilities of NGS to investigate the reported association between glioblastoma multiforme (GBM) and human cytomegalovirus (HCMV). A large-scale comprehensive virome assessment was performed on publicly available sequencing datasets from the Cancer Genome Atlas (TCGA), including RNA-seq datasets from primary GBM (n = 157), recurrent GBM (n = 13), low-grade gliomas (n = 514), recurrent low-grade gliomas (n = 17), and normal brain (n = 5), and whole genome sequencing (WGS) datasets from primary GBM (n = 51), recurrent GBM (n = 10), and normal matched blood samples (n = 20). In addition, RNA-seq datasets from MRI-guided biopsies (n = 92) and glioma stem-like cell cultures (n = 9) were analyzed. Sixty-four DNA-seq datasets from 11 meningiomas and their corresponding blood control samples were also analyzed. Finally, three primary GBM tissue samples were obtained, sequenced using RNA-seq, and analyzed. After in-depth analysis, the most robust virus findings were the detection of papillomavirus (HPV) and hepatitis B reads in the occasional LGG sample (4 samples and 1 sample, respectively). In addition, low numbers of virus reads were detected in several datasets but detailed investigation of these reads suggest that these findings likely represent artifacts or non-pathological infections. For example, all of the sporadic low level HCMV reads were found to map to the immediate early promoter intimating that they likely originated from laboratory expression vector contamination. Despite the detection of low numbers of Epstein-Barr virus reads in some samples, these likely originated from infiltrating B-cells. Finally, human herpesvirus 6 and 7 aligned viral reads were identified in all DNA-seq and a few RNA-seq datasets but detailed analysis demonstrated that these were likely derived from the homologous human telomeric-like repeats. Other low abundance viral reads were detected in some samples but for most viruses, the reads likely represent artifacts or incidental infections. This analysis argues against associations between most known viruses and GBM or mengiomas. Nevertheless, there may be a low percentage association between HPV and/or hepatitis B and LGGs.

No MeSH data available.


Related in: MedlinePlus

Heat map showing the number of viral reads per million human mapped (RPHM) reads for brain tissue RNA-seq datasets. Color intensity represents relative viral RPHM across all datasets
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4940872&req=5

Fig1: Heat map showing the number of viral reads per million human mapped (RPHM) reads for brain tissue RNA-seq datasets. Color intensity represents relative viral RPHM across all datasets

Mentions: Previous studies have used human read subtraction-based approaches to assess the metatranscriptomic profile of primary and recurrent glioma RNA-seq datasets from The Cancer Genome Atlas (TCGA) where a lack of association with HCMV was reported [10, 14, 26, 27]. To investigate this issue further, we first performed a global virome analysis on these samples (as well as 5 normal brain tissue samples), using a more directed, non-subtraction based approach that we have reported previously [33, 50, 53]. For this analysis, we directly aligned all reads from these datasets to an alignment index containing the human genome plus a library of 740 virus genomes (including sequences from all known human viruses documented by NCBI) using the Spliced Transcripts Alignment to a Reference (STAR) aligner. Running this virome pipeline on a known EBV-associated gastric cancer tissue biopsy cohort from TCGA [54] showed EBV read levels which ranged from 7–400 viral Reads Per Mapped Human reads (RPMH) [53]. Like PCR, next generation sequencing is susceptible to low level contamination issues [34]. Nevertheless, in an attempt to capture potential low abundance infections, we set a viral read threshold that was 10 times lower than the lowest viral RPMH value for EBV in the gastric cancer cohort [53, 54]. Based on this requirement of at least 0.7 viral RPMH, no virus associations were called in 157 primary and 13 recurrent gliomas or in 5 normal brain RNA-seq datasets analyzed except for the finding of 2.1 human herpesvirus (HHV) -6 or HHV-7 RPMH (note: HHV-6 and HHV-7 reads are considered together here due to all reads mapping to a homologous region of these two viruses) in one recurrent glioma (Fig. 1, Additional file 3: File S1). Notably, all HHV6/7 reads in this sample were found to contain the simple sequence, TAACCC, a repeat found in both of these virus genomes and in human telomeric repeats. Since no HHV6/7 assigned reads mapped outside of the TAACCC repeat region of the viral genomes, we conclude that these reads likely originated from contaminating genomic sequences from human telomeric repeats.Fig. 1


A comprehensive next generation sequencing-based virome assessment in brain tissue suggests no major virus - tumor association.

Strong MJ, Blanchard E, Lin Z, Morris CA, Baddoo M, Taylor CM, Ware ML, Flemington EK - Acta Neuropathol Commun (2016)

Heat map showing the number of viral reads per million human mapped (RPHM) reads for brain tissue RNA-seq datasets. Color intensity represents relative viral RPHM across all datasets
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940872&req=5

Fig1: Heat map showing the number of viral reads per million human mapped (RPHM) reads for brain tissue RNA-seq datasets. Color intensity represents relative viral RPHM across all datasets
Mentions: Previous studies have used human read subtraction-based approaches to assess the metatranscriptomic profile of primary and recurrent glioma RNA-seq datasets from The Cancer Genome Atlas (TCGA) where a lack of association with HCMV was reported [10, 14, 26, 27]. To investigate this issue further, we first performed a global virome analysis on these samples (as well as 5 normal brain tissue samples), using a more directed, non-subtraction based approach that we have reported previously [33, 50, 53]. For this analysis, we directly aligned all reads from these datasets to an alignment index containing the human genome plus a library of 740 virus genomes (including sequences from all known human viruses documented by NCBI) using the Spliced Transcripts Alignment to a Reference (STAR) aligner. Running this virome pipeline on a known EBV-associated gastric cancer tissue biopsy cohort from TCGA [54] showed EBV read levels which ranged from 7–400 viral Reads Per Mapped Human reads (RPMH) [53]. Like PCR, next generation sequencing is susceptible to low level contamination issues [34]. Nevertheless, in an attempt to capture potential low abundance infections, we set a viral read threshold that was 10 times lower than the lowest viral RPMH value for EBV in the gastric cancer cohort [53, 54]. Based on this requirement of at least 0.7 viral RPMH, no virus associations were called in 157 primary and 13 recurrent gliomas or in 5 normal brain RNA-seq datasets analyzed except for the finding of 2.1 human herpesvirus (HHV) -6 or HHV-7 RPMH (note: HHV-6 and HHV-7 reads are considered together here due to all reads mapping to a homologous region of these two viruses) in one recurrent glioma (Fig. 1, Additional file 3: File S1). Notably, all HHV6/7 reads in this sample were found to contain the simple sequence, TAACCC, a repeat found in both of these virus genomes and in human telomeric repeats. Since no HHV6/7 assigned reads mapped outside of the TAACCC repeat region of the viral genomes, we conclude that these reads likely originated from contaminating genomic sequences from human telomeric repeats.Fig. 1

Bottom Line: For example, all of the sporadic low level HCMV reads were found to map to the immediate early promoter intimating that they likely originated from laboratory expression vector contamination.This analysis argues against associations between most known viruses and GBM or mengiomas.Nevertheless, there may be a low percentage association between HPV and/or hepatitis B and LGGs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Tulane University, New Orleans, LA, 70112, USA.

ABSTRACT
Next generation sequencing (NGS) can globally interrogate the genetic composition of biological samples in an unbiased yet sensitive manner. The objective of this study was to utilize the capabilities of NGS to investigate the reported association between glioblastoma multiforme (GBM) and human cytomegalovirus (HCMV). A large-scale comprehensive virome assessment was performed on publicly available sequencing datasets from the Cancer Genome Atlas (TCGA), including RNA-seq datasets from primary GBM (n = 157), recurrent GBM (n = 13), low-grade gliomas (n = 514), recurrent low-grade gliomas (n = 17), and normal brain (n = 5), and whole genome sequencing (WGS) datasets from primary GBM (n = 51), recurrent GBM (n = 10), and normal matched blood samples (n = 20). In addition, RNA-seq datasets from MRI-guided biopsies (n = 92) and glioma stem-like cell cultures (n = 9) were analyzed. Sixty-four DNA-seq datasets from 11 meningiomas and their corresponding blood control samples were also analyzed. Finally, three primary GBM tissue samples were obtained, sequenced using RNA-seq, and analyzed. After in-depth analysis, the most robust virus findings were the detection of papillomavirus (HPV) and hepatitis B reads in the occasional LGG sample (4 samples and 1 sample, respectively). In addition, low numbers of virus reads were detected in several datasets but detailed investigation of these reads suggest that these findings likely represent artifacts or non-pathological infections. For example, all of the sporadic low level HCMV reads were found to map to the immediate early promoter intimating that they likely originated from laboratory expression vector contamination. Despite the detection of low numbers of Epstein-Barr virus reads in some samples, these likely originated from infiltrating B-cells. Finally, human herpesvirus 6 and 7 aligned viral reads were identified in all DNA-seq and a few RNA-seq datasets but detailed analysis demonstrated that these were likely derived from the homologous human telomeric-like repeats. Other low abundance viral reads were detected in some samples but for most viruses, the reads likely represent artifacts or incidental infections. This analysis argues against associations between most known viruses and GBM or mengiomas. Nevertheless, there may be a low percentage association between HPV and/or hepatitis B and LGGs.

No MeSH data available.


Related in: MedlinePlus