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Low-cost, low-input RNA-seq protocols perform nearly as well as high-input protocols.

Combs PA, Eisen MB - PeerJ (2015)

Bottom Line: Recently, a number of protocols extending RNA-sequencing to the single-cell regime have been published.In this study, we performed a critical evaluation of several of these low-volume RNA-seq protocols, and found that they performed slightly less well in per-gene linearity of response, but with at least two orders of magnitude less sample required.We also explored a simple modification to one of these protocols that, for many samples, reduced the cost of library preparation to approximately $20/sample.

View Article: PubMed Central - HTML - PubMed

Affiliation: Graduate Program in Biophysics, University of California , Berkeley, CA , USA.

ABSTRACT
Recently, a number of protocols extending RNA-sequencing to the single-cell regime have been published. However, we were concerned that the additional steps to deal with such minute quantities of input sample would introduce serious biases that would make analysis of the data using existing approaches invalid. In this study, we performed a critical evaluation of several of these low-volume RNA-seq protocols, and found that they performed slightly less well in per-gene linearity of response, but with at least two orders of magnitude less sample required. We also explored a simple modification to one of these protocols that, for many samples, reduced the cost of library preparation to approximately $20/sample.

No MeSH data available.


Distributions of slopes, intercepts, and correlation coefficients for experiment 3.Nextera XT reactions were reduced in volume by the indicated amount.
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fig-2: Distributions of slopes, intercepts, and correlation coefficients for experiment 3.Nextera XT reactions were reduced in volume by the indicated amount.

Mentions: When normalized to the same number of reads as in experiment 2, the protocols with diluted Nextera reagents performed effectively identically: for instance, the mean correlation coefficients were in both cases 0.96 ± 0.05 (Fig. 2 and Table 4). This is despite the additional cycles of enrichment, which improved yield.


Low-cost, low-input RNA-seq protocols perform nearly as well as high-input protocols.

Combs PA, Eisen MB - PeerJ (2015)

Distributions of slopes, intercepts, and correlation coefficients for experiment 3.Nextera XT reactions were reduced in volume by the indicated amount.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380159&req=5

fig-2: Distributions of slopes, intercepts, and correlation coefficients for experiment 3.Nextera XT reactions were reduced in volume by the indicated amount.
Mentions: When normalized to the same number of reads as in experiment 2, the protocols with diluted Nextera reagents performed effectively identically: for instance, the mean correlation coefficients were in both cases 0.96 ± 0.05 (Fig. 2 and Table 4). This is despite the additional cycles of enrichment, which improved yield.

Bottom Line: Recently, a number of protocols extending RNA-sequencing to the single-cell regime have been published.In this study, we performed a critical evaluation of several of these low-volume RNA-seq protocols, and found that they performed slightly less well in per-gene linearity of response, but with at least two orders of magnitude less sample required.We also explored a simple modification to one of these protocols that, for many samples, reduced the cost of library preparation to approximately $20/sample.

View Article: PubMed Central - HTML - PubMed

Affiliation: Graduate Program in Biophysics, University of California , Berkeley, CA , USA.

ABSTRACT
Recently, a number of protocols extending RNA-sequencing to the single-cell regime have been published. However, we were concerned that the additional steps to deal with such minute quantities of input sample would introduce serious biases that would make analysis of the data using existing approaches invalid. In this study, we performed a critical evaluation of several of these low-volume RNA-seq protocols, and found that they performed slightly less well in per-gene linearity of response, but with at least two orders of magnitude less sample required. We also explored a simple modification to one of these protocols that, for many samples, reduced the cost of library preparation to approximately $20/sample.

No MeSH data available.