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A combination of LongSAGE with Solexa sequencing is well suited to explore the depth and the complexity of transcriptome.

Hanriot L, Keime C, Gay N, Faure C, Dossat C, Wincker P, Scoté-Blachon C, Peyron C, Gandrillon O - BMC Genomics (2008)

Bottom Line: Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript.However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered.We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method.

View Article: PubMed Central - HTML - PubMed

Affiliation: UMR5534 CNRS Université Claude Bernard Lyon1, Université de Lyon, Institut Fédératif des Neurosciences de Lyon, Lyon cedex, France. lucie.hanriot@hotmail.fr

ABSTRACT

Background: "Open" transcriptome analysis methods allow to study gene expression without a priori knowledge of the transcript sequences. As of now, SAGE (Serial Analysis of Gene Expression), LongSAGE and MPSS (Massively Parallel Signature Sequencing) are the mostly used methods for "open" transcriptome analysis. Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript. In contrast to LongSAGE, the high throughput sequencing method used in MPSS enables the rapid sequencing of very large libraries containing several millions of tags, allowing deep transcriptome analysis. However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered.

Results: In order to make a deep analysis of mouse hypothalamus transcriptome avoiding the limitation introduced by MPSS, we combined LongSAGE with the Solexa sequencing technology and obtained a library of more than 11 millions of tags. We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method.

Conclusion: We found that Solexa sequencing technology combined with LongSAGE is perfectly suited for deep transcriptome analysis. In contrast to MPSS, it gives a complex representation of transcriptome as reliable as a LongSAGE library sequenced by the Sanger method.

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Related in: MedlinePlus

Expression level for 3 well-known genes of the hypothalamus, using three different techniques. The level of expression of three genes (pro-melanin concentrating hormone (Pmch), preprohypocretin (Hcrt) and prodynorphin (Pdyn)) known to be expressed in the hypothalamus is evaluated as their number of occurrence from the Sanger_Hypo library (darkest bars) and the Solexa_Hypo library (lighest bars) as tags per million (left axis). The level of expression of these 3 genes is also evaluated from 6 independent hypothalamic samples by qPCR (right axis). The mean and standard deviation are reported in copies of transcripts (right axis).
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Figure 3: Expression level for 3 well-known genes of the hypothalamus, using three different techniques. The level of expression of three genes (pro-melanin concentrating hormone (Pmch), preprohypocretin (Hcrt) and prodynorphin (Pdyn)) known to be expressed in the hypothalamus is evaluated as their number of occurrence from the Sanger_Hypo library (darkest bars) and the Solexa_Hypo library (lighest bars) as tags per million (left axis). The level of expression of these 3 genes is also evaluated from 6 independent hypothalamic samples by qPCR (right axis). The mean and standard deviation are reported in copies of transcripts (right axis).

Mentions: To validate the procedure of tissue collection, we looked at genes known to be absent in the hypothalamus but expressed in adjacent brain area such as the thalamus or the midbrain. Location was verified using the Allen Brain atlas of gene expression in mouse [18]. We selected the NMDA NR2C, the chloride channel calcium activated 2 and the sodium voltage gated type V alpha. None of them were found in the Sanger_Hypo or the Solexa_Hypo libraries. As libraries were constructed from two different hypothalamic samples, two different strains of mice and an amplification step was added for the built-up of the Solexa_Hypo library (Table 1), a direct comparison of the level of expression of selected genes is meaningless. We nevertheless checked for the expression level of 3 well-known genes of the hypothalamus, the pro-melanin concentrating hormone (Pmch), preprohypocretin (Hcrt) and prodynorphin (Pdyn) and found that in both libraries (Sanger_Hypo and Solexa_Hypo) and with q-PCR, the level of expression of Pmch is remarkably higher than Hcrt that is greatly elevated compare to Pdyn (Figure 3). This demonstrates the overall agreement between those three techniques.


A combination of LongSAGE with Solexa sequencing is well suited to explore the depth and the complexity of transcriptome.

Hanriot L, Keime C, Gay N, Faure C, Dossat C, Wincker P, Scoté-Blachon C, Peyron C, Gandrillon O - BMC Genomics (2008)

Expression level for 3 well-known genes of the hypothalamus, using three different techniques. The level of expression of three genes (pro-melanin concentrating hormone (Pmch), preprohypocretin (Hcrt) and prodynorphin (Pdyn)) known to be expressed in the hypothalamus is evaluated as their number of occurrence from the Sanger_Hypo library (darkest bars) and the Solexa_Hypo library (lighest bars) as tags per million (left axis). The level of expression of these 3 genes is also evaluated from 6 independent hypothalamic samples by qPCR (right axis). The mean and standard deviation are reported in copies of transcripts (right axis).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562395&req=5

Figure 3: Expression level for 3 well-known genes of the hypothalamus, using three different techniques. The level of expression of three genes (pro-melanin concentrating hormone (Pmch), preprohypocretin (Hcrt) and prodynorphin (Pdyn)) known to be expressed in the hypothalamus is evaluated as their number of occurrence from the Sanger_Hypo library (darkest bars) and the Solexa_Hypo library (lighest bars) as tags per million (left axis). The level of expression of these 3 genes is also evaluated from 6 independent hypothalamic samples by qPCR (right axis). The mean and standard deviation are reported in copies of transcripts (right axis).
Mentions: To validate the procedure of tissue collection, we looked at genes known to be absent in the hypothalamus but expressed in adjacent brain area such as the thalamus or the midbrain. Location was verified using the Allen Brain atlas of gene expression in mouse [18]. We selected the NMDA NR2C, the chloride channel calcium activated 2 and the sodium voltage gated type V alpha. None of them were found in the Sanger_Hypo or the Solexa_Hypo libraries. As libraries were constructed from two different hypothalamic samples, two different strains of mice and an amplification step was added for the built-up of the Solexa_Hypo library (Table 1), a direct comparison of the level of expression of selected genes is meaningless. We nevertheless checked for the expression level of 3 well-known genes of the hypothalamus, the pro-melanin concentrating hormone (Pmch), preprohypocretin (Hcrt) and prodynorphin (Pdyn) and found that in both libraries (Sanger_Hypo and Solexa_Hypo) and with q-PCR, the level of expression of Pmch is remarkably higher than Hcrt that is greatly elevated compare to Pdyn (Figure 3). This demonstrates the overall agreement between those three techniques.

Bottom Line: Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript.However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered.We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method.

View Article: PubMed Central - HTML - PubMed

Affiliation: UMR5534 CNRS Université Claude Bernard Lyon1, Université de Lyon, Institut Fédératif des Neurosciences de Lyon, Lyon cedex, France. lucie.hanriot@hotmail.fr

ABSTRACT

Background: "Open" transcriptome analysis methods allow to study gene expression without a priori knowledge of the transcript sequences. As of now, SAGE (Serial Analysis of Gene Expression), LongSAGE and MPSS (Massively Parallel Signature Sequencing) are the mostly used methods for "open" transcriptome analysis. Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript. In contrast to LongSAGE, the high throughput sequencing method used in MPSS enables the rapid sequencing of very large libraries containing several millions of tags, allowing deep transcriptome analysis. However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered.

Results: In order to make a deep analysis of mouse hypothalamus transcriptome avoiding the limitation introduced by MPSS, we combined LongSAGE with the Solexa sequencing technology and obtained a library of more than 11 millions of tags. We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method.

Conclusion: We found that Solexa sequencing technology combined with LongSAGE is perfectly suited for deep transcriptome analysis. In contrast to MPSS, it gives a complex representation of transcriptome as reliable as a LongSAGE library sequenced by the Sanger method.

Show MeSH
Related in: MedlinePlus