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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

Number of transcripts detected at increased sampling rate. In silico analysis using the sum of aligned reads (total number of reads ~60 million) across each group (reduced, non-reduced). A random subset of these aligned reads is then used to find the number of detected transcripts across the two sample groups over the 5 tag, 5 out of 6 samples threshold. The plot shows that at similar sequencing depths, there is a consistently higher number of transcripts detected in the globin reduced samples compared to the non-globin reduced samples.
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Figure 5: Number of transcripts detected at increased sampling rate. In silico analysis using the sum of aligned reads (total number of reads ~60 million) across each group (reduced, non-reduced). A random subset of these aligned reads is then used to find the number of detected transcripts across the two sample groups over the 5 tag, 5 out of 6 samples threshold. The plot shows that at similar sequencing depths, there is a consistently higher number of transcripts detected in the globin reduced samples compared to the non-globin reduced samples.

Mentions: The previous results showed that by reducing the number of globin transcripts before sequencing, the number of different transcripts that can be identified increases. Next, we investigated how much additional sequencing is required to achieve this effect of globin reduction just by sequencing non-globin reduced samples at a greater depth. For this purpose we compared results from non-globin reduced RNA samples that were sequenced at high and low sequencing depth. At two times higher sequencing depth, the reported alignments as well as the number of identified transcripts for each of the samples increased to the level of the globin reduced samples (Table 4). The number of 13,000 detected transcripts in globin-reduced samples is achieved at a sequencing depth of ~15 million reads while the same number of transcripts is detected at a sequencing depth of ~25 million reads in the non-globin reduced samples. In silico analysis was performed to determine at which sampling rate the threshold of at least 5 tags in 5 out the 6 samples was reached. The difference in detected transcripts between the reduced and non-reduced samples at any given sequencing depth remained equal, indicating that even at a very large numbers of reads (> 60 million reads) whole blood samples are not sequenced to saturation (see Figure 5).


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)

Number of transcripts detected at increased sampling rate. In silico analysis using the sum of aligned reads (total number of reads ~60 million) across each group (reduced, non-reduced). A random subset of these aligned reads is then used to find the number of detected transcripts across the two sample groups over the 5 tag, 5 out of 6 samples threshold. The plot shows that at similar sequencing depths, there is a consistently higher number of transcripts detected in the globin reduced samples compared to the non-globin reduced samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Number of transcripts detected at increased sampling rate. In silico analysis using the sum of aligned reads (total number of reads ~60 million) across each group (reduced, non-reduced). A random subset of these aligned reads is then used to find the number of detected transcripts across the two sample groups over the 5 tag, 5 out of 6 samples threshold. The plot shows that at similar sequencing depths, there is a consistently higher number of transcripts detected in the globin reduced samples compared to the non-globin reduced samples.
Mentions: The previous results showed that by reducing the number of globin transcripts before sequencing, the number of different transcripts that can be identified increases. Next, we investigated how much additional sequencing is required to achieve this effect of globin reduction just by sequencing non-globin reduced samples at a greater depth. For this purpose we compared results from non-globin reduced RNA samples that were sequenced at high and low sequencing depth. At two times higher sequencing depth, the reported alignments as well as the number of identified transcripts for each of the samples increased to the level of the globin reduced samples (Table 4). The number of 13,000 detected transcripts in globin-reduced samples is achieved at a sequencing depth of ~15 million reads while the same number of transcripts is detected at a sequencing depth of ~25 million reads in the non-globin reduced samples. In silico analysis was performed to determine at which sampling rate the threshold of at least 5 tags in 5 out the 6 samples was reached. The difference in detected transcripts between the reduced and non-reduced samples at any given sequencing depth remained equal, indicating that even at a very large numbers of reads (> 60 million reads) whole blood samples are not sequenced to saturation (see Figure 5).

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