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Bulk RNA-seq analysis

Ngày 27 tháng 09 năm 2026

TS. Lưu Phúc Lợi

Email: luu.p.loi@googlemail.com

Zalo: 0901802182

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Content

  • What is RNA-seq?
  • Why study RNA?
  • Different Types of RNA – Different Library Preps

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What is RNA-seq?

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From BAM to Count

Gene

Sample 1 Healthy

Sample 2 Healthy

Sample 3 Healthy

​

Sample 4 Tumor

​

Sample 5 Tumor

​

Sample 6 Tumor

A

100

101

105

160

163

154

B

10

8

9

1

2

3

C

45

45

45

46

46

46

D

0.11

0.12

0.13

0.0012

0.0014

0.0013

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Differential Gene Expression

Gene

Sample 1 Healthy

Sample 2 Healthy

Sample 3 Healthy

​

Sample 4 Tumor

​

Sample 5 Tumor

​

Sample 6 Tumor

A

100

101

105

160

163

154

B

10

8

9

1

2

3

C

45

45

45

46

46

46

D

0.11

0.12

0.13

0.0012

0.0014

0.0013

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Why study RNA?

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Different Types of RNA – Different Library Preps

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Direct 3' and 5' adapter ligation (C) is sub-optimal for differential expression analysis primarily due to severe sequence-dependent ligation bias and technical artifacts:

  • RNA Ligase Preference: The enzymes used (e.g., T4 RNA ligase) show strong preferences for specific primary sequences and secondary structures formed at the junction between the adapter and the short RNA molecule. Some small RNA species are ligated with high efficiency, while others are barely ligated at all.
  • Distortion of Relative Abundances: Because ligation efficiency varies drastically (up to tens or hundreds-fold) between different sequences, the final read counts do not accurately reflect the actual stoichiometry/abundance of the small RNAs in the biological sample.
  • Formation of Adapter Dimers: Direct sequential ligation can easily form adapter-adapter dimers without an insert, competing for sequencing reads and lowering overall library complexity unless rigorous gel/bead size selection is applied.
  • Length Constraints: Short read lengths <100bp and strict adapter constraints limit the ability to map reads uniquely across repetitive loci or distinguish highly similar gene isoforms.

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Cảm ơn sự lắng nghe của các bạn!��Lưu Phúc Lợi, PhD�Zalo: 0901802182�Email: luu.p.loi@googlemail.com