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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

Illustration of the extent of tissue collection. A: Photograph of a frontal 400 μm-thick section of a mouse brain at the level of the hypothalamus. The hypothalamus, centered on the perifornical nucleus was collected bilaterally using a trocard of 1 mm diameter. Scale bar = 1 mm. B: Schematic drawing of the section presented in A and extracted from the mouse atlas of G Paxinos & KB Franklin, (+1,98 mm interaural). The blue circle highlights the extent of the brain area taken off. 3V: third ventricule; Arc: arcuate nucleus; CM: centro-medial thalamic nucleus; cp: cerebral pedoncule; DMH: dorsomedial hypothalamic nucleus; f: fornix; ic: internal capsule; LH: lateral hypothalamic area, ml: median lemniscus; mt: mammillothalamic tract; opt: optic tract; PeF: perifornical nucleus; PH: posterior hypothalamic area; Re: thalamic reuniens nucleus; st: stria terminalis; VMH: ventromedial hypothalamic nucleus.
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Figure 2: Illustration of the extent of tissue collection. A: Photograph of a frontal 400 μm-thick section of a mouse brain at the level of the hypothalamus. The hypothalamus, centered on the perifornical nucleus was collected bilaterally using a trocard of 1 mm diameter. Scale bar = 1 mm. B: Schematic drawing of the section presented in A and extracted from the mouse atlas of G Paxinos & KB Franklin, (+1,98 mm interaural). The blue circle highlights the extent of the brain area taken off. 3V: third ventricule; Arc: arcuate nucleus; CM: centro-medial thalamic nucleus; cp: cerebral pedoncule; DMH: dorsomedial hypothalamic nucleus; f: fornix; ic: internal capsule; LH: lateral hypothalamic area, ml: median lemniscus; mt: mammillothalamic tract; opt: optic tract; PeF: perifornical nucleus; PH: posterior hypothalamic area; Re: thalamic reuniens nucleus; st: stria terminalis; VMH: ventromedial hypothalamic nucleus.

Mentions: Two libraries were created from male adult mice hypothalamus, collected bilaterally as punches centered on the perifornical nucleus from the caudal part of the paraventricular nucleus to the mammillary bodies (Figure 2). A first library was constructed by using the LongSAGE method combined with the Sanger sequencing technology (Sanger_Hypo), and a second library with the LongSAGE method combined with the Solexa sequencing technology (Solexa_Hypo) (Figure 1). Major characteristics of both libraries are summarized in Table 1.


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)

Illustration of the extent of tissue collection. A: Photograph of a frontal 400 μm-thick section of a mouse brain at the level of the hypothalamus. The hypothalamus, centered on the perifornical nucleus was collected bilaterally using a trocard of 1 mm diameter. Scale bar = 1 mm. B: Schematic drawing of the section presented in A and extracted from the mouse atlas of G Paxinos & KB Franklin, (+1,98 mm interaural). The blue circle highlights the extent of the brain area taken off. 3V: third ventricule; Arc: arcuate nucleus; CM: centro-medial thalamic nucleus; cp: cerebral pedoncule; DMH: dorsomedial hypothalamic nucleus; f: fornix; ic: internal capsule; LH: lateral hypothalamic area, ml: median lemniscus; mt: mammillothalamic tract; opt: optic tract; PeF: perifornical nucleus; PH: posterior hypothalamic area; Re: thalamic reuniens nucleus; st: stria terminalis; VMH: ventromedial hypothalamic nucleus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Illustration of the extent of tissue collection. A: Photograph of a frontal 400 μm-thick section of a mouse brain at the level of the hypothalamus. The hypothalamus, centered on the perifornical nucleus was collected bilaterally using a trocard of 1 mm diameter. Scale bar = 1 mm. B: Schematic drawing of the section presented in A and extracted from the mouse atlas of G Paxinos & KB Franklin, (+1,98 mm interaural). The blue circle highlights the extent of the brain area taken off. 3V: third ventricule; Arc: arcuate nucleus; CM: centro-medial thalamic nucleus; cp: cerebral pedoncule; DMH: dorsomedial hypothalamic nucleus; f: fornix; ic: internal capsule; LH: lateral hypothalamic area, ml: median lemniscus; mt: mammillothalamic tract; opt: optic tract; PeF: perifornical nucleus; PH: posterior hypothalamic area; Re: thalamic reuniens nucleus; st: stria terminalis; VMH: ventromedial hypothalamic nucleus.
Mentions: Two libraries were created from male adult mice hypothalamus, collected bilaterally as punches centered on the perifornical nucleus from the caudal part of the paraventricular nucleus to the mammillary bodies (Figure 2). A first library was constructed by using the LongSAGE method combined with the Sanger sequencing technology (Sanger_Hypo), and a second library with the LongSAGE method combined with the Solexa sequencing technology (Solexa_Hypo) (Figure 1). Major characteristics of both libraries are summarized in Table 1.

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