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Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA.

Mastrokolias A, den Dunnen JT, van Ommen GB, 't Hoen PA, van Roon-Mom WM - BMC Genomics (2012)

Bottom Line: Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples.In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples.The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Human and Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, 2333ZC, Leiden, The Netherlands.

ABSTRACT

Background: Transcriptome analysis is of great interest in clinical research, where significant differences between individuals can be translated into biomarkers of disease. Although next generation sequencing provides robust, comparable and highly informative expression profiling data, with several million of tags per blood sample, reticulocyte globin transcripts can constitute up to 76% of total mRNA compromising the detection of low abundant transcripts. We have removed globin transcripts from 6 human whole blood RNA samples with a human globin reduction kit and compared them with the same non-reduced samples using deep Serial Analysis of Gene Expression.

Results: Globin tags comprised 52-76% of total tags in our samples. Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples. In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples. Removing globin transcripts allowed us to also identify 2112 genes that could not be detected in the non-globin reduced samples, with roles in cell surface receptor signal transduction, G-protein coupled receptor protein signalling pathways and neurological processes.

Conclusions: The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

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Related in: MedlinePlus

Venn diagram displaying the number of transcripts in at least 5 out of 6 samples at a level of 5 tags or more. Reducing the globin transcripts increases the number of transcripts that can be reliably detected (2112 genes). 9121 genes were not consistently expressed in either group.
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Figure 3: Venn diagram displaying the number of transcripts in at least 5 out of 6 samples at a level of 5 tags or more. Reducing the globin transcripts increases the number of transcripts that can be reliably detected (2112 genes). 9121 genes were not consistently expressed in either group.

Mentions: From the total number of 21,633 transcripts discovered in our SAGE experiments, 10,368 were expressed above the threshold of at least 5 tags in 5 out of the 6 samples in both the globin reduced and non-reduced group. We identified 2,112 transcripts in the globin-reduced samples that were not expressed above the threshold in the non-reduced samples and 32 transcripts in the non-globin reduced samples that could not be detected in the globin-reduced samples (see Figure 3). Additionally, 9,121 transcripts were detected in globin-reduced samples only but did not reach the threshold (low abundance transcripts). Increasing the threshold tag number resulted in a higher number of genes detected by the globin reduced samples while the number of genes only detected in non-reduced samples remained approximately the same. Furthermore increasing the threshold resulted in less genes identified by both groups.


Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA.

Mastrokolias A, den Dunnen JT, van Ommen GB, 't Hoen PA, van Roon-Mom WM - BMC Genomics (2012)

Venn diagram displaying the number of transcripts in at least 5 out of 6 samples at a level of 5 tags or more. Reducing the globin transcripts increases the number of transcripts that can be reliably detected (2112 genes). 9121 genes were not consistently expressed in either group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275489&req=5

Figure 3: Venn diagram displaying the number of transcripts in at least 5 out of 6 samples at a level of 5 tags or more. Reducing the globin transcripts increases the number of transcripts that can be reliably detected (2112 genes). 9121 genes were not consistently expressed in either group.
Mentions: From the total number of 21,633 transcripts discovered in our SAGE experiments, 10,368 were expressed above the threshold of at least 5 tags in 5 out of the 6 samples in both the globin reduced and non-reduced group. We identified 2,112 transcripts in the globin-reduced samples that were not expressed above the threshold in the non-reduced samples and 32 transcripts in the non-globin reduced samples that could not be detected in the globin-reduced samples (see Figure 3). Additionally, 9,121 transcripts were detected in globin-reduced samples only but did not reach the threshold (low abundance transcripts). Increasing the threshold tag number resulted in a higher number of genes detected by the globin reduced samples while the number of genes only detected in non-reduced samples remained approximately the same. Furthermore increasing the threshold resulted in less genes identified by both groups.

Bottom Line: Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples.In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples.The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Human and Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, 2333ZC, Leiden, The Netherlands.

ABSTRACT

Background: Transcriptome analysis is of great interest in clinical research, where significant differences between individuals can be translated into biomarkers of disease. Although next generation sequencing provides robust, comparable and highly informative expression profiling data, with several million of tags per blood sample, reticulocyte globin transcripts can constitute up to 76% of total mRNA compromising the detection of low abundant transcripts. We have removed globin transcripts from 6 human whole blood RNA samples with a human globin reduction kit and compared them with the same non-reduced samples using deep Serial Analysis of Gene Expression.

Results: Globin tags comprised 52-76% of total tags in our samples. Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples. In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples. Removing globin transcripts allowed us to also identify 2112 genes that could not be detected in the non-globin reduced samples, with roles in cell surface receptor signal transduction, G-protein coupled receptor protein signalling pathways and neurological processes.

Conclusions: The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

Show MeSH
Related in: MedlinePlus