Single-cell sequencing, three groups, not the main body of the experiment, is it possible to have one sample per group?

In single-cell sequencing experiments, designing with only one sample per group is feasible but there are some issues to be aware of:

1. Insufficient statistical confidence

Having one sample per group means there are no biological replicates. Although single-cell sequencing generates large amounts of data and the variation between cells can partially reflect biological differences, relying entirely on a single sample can lead to the following issues:

(1) Inability to assess biological variation: The heterogeneity within a sample's cells does not equate to true biological differences between groups.

(2) Statistical instability: The differential expression genes or other results obtained might just be a reflection of random noise.

 

2. Limitations in interpreting experimental conclusions

(1) When analyzing results, it is difficult to clearly attribute inter-group differences to biological phenomena rather than random or technical biases (batch effects).

(2) If the focus of the experiment is on preliminary exploration of certain trends rather than rigorous validation, a single sample design can serve as a reference attempt.

 

3. Impact of technical variation

Single-cell sequencing involves complex sample handling and library construction processes, each of which may introduce technical biases. Without replicate samples, it is difficult to distinguish whether the differences between experimental groups stem from technical variation.

 

I. When is a single sample acceptable?

1. Exploratory experiments: If the aim of the study is exploratory analysis, such as verifying the feasibility of a certain experimental method, a single sample design can serve as a trial scheme.

2. Rare samples: If certain samples are very rare or difficult to obtain (such as tissue samples from patients with rare diseases), a single sample is also a reasonable choice.

3. Subsequent validation: Preliminary results from single-sample sequencing can be further validated using other experimental methods (such as qPCR, flow cytometry).

 

II. Suggestions for improvement

1. Increase biological replicates: Preparing at least 3 biological replicates per group is a common minimum standard for more reliably assessing inter-group differences.

2. Balance cost and sample size appropriately: If cost constraints are significant, the depth of single-cell sequencing can be reduced or target genes can be screened to accommodate more samples.

3. Subsequent validation: If only single-sample sequencing can be conducted, it is recommended to validate key findings through independent experiments (such as RT-qPCR, immunohistochemistry) to enhance the credibility of conclusions.

 

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