December 13, 2021 [reading notes] | understanding of chain specific database building

When I first came into contact with high-throughput sequencing, I knew the concept of chain specific library building , At that time, we also knew that we could make use of Jia U Law , But I didn't think about the details . Recently, I broke up this concept and thoroughly understood it , Finally filled the hole .

Before the formal presentation , There are a few concepts to be clear about .

DNA The positive and negative chains of , It is the two opposite complementary chains . The chain given by the reference genome is the so-called positive chain （forword）, The other chain is the anti chain （reverse）. But the pros and cons must not be linked to the chain of justice （sense strand） Antisense chain （antisense strand） confusion .

Justice chain （sense strand）： Two complementary DNA One of the chains that carries the information encoding the protein is called the justice chain , Also known as coding chain , Because its sequence is similar to mRNA identical .

Antisense chain （antisense strand）： Another complementary chain is called the antisense chain . While the antisense chain is similar to RNA Reverse complementarity , But it really gives RNA When the template chain , So the antisense chain is also a template chain .

It should be noted that ： In a double strand containing several genes DNA In molecules , The justice chains of all genes are not all on the same chain .

in other words , The justice chain of some genes is positive （forword strand）, The justice chain of some genes is anti chain （reverse strand）. therefore ,DNA One of the two chains is a justice chain for some genes , For other genes, it is an antisense chain

Sum up two points

Justice chain （sense strand）= Coding chain （coding strand）= Non template chain

forword strand There can be both sense strand and antisense strand. Because these are two different concepts

Let's take a look at the common... Through this schematic diagram of database building RNA-Seq What is the difference between building a library and building a chain specific library

First of all, let's talk about ordinary RNA-Seq How to build database ： It's in RNA Reverse transcription into double chains cDNA Both ends , Two are added symmetrically Y Type connector , Then it becomes a library . It has a drawback , Is that it is a double chain DNA sequencing . So after measuring the sequence , We don't know what we're measuring reads Is it from the positive chain or the negative chain .

And chain specific library building （ In the middle of the picture dUTP Methods as an example ） First, random primers are used to synthesize RNA One of cDNA chain , In the synthesis of the second chain dUTP Instead of dTTP, Add adaptor After use UDGase Handle , There will be U The second one cDNA Degradation . After degradation occurs , The double chain library has only one chain left （ Negative chain ）. And the two ends of this chain are the joints of different sequences . adopt PCR amplification , In the end, only the first one was retained cDNA（ Negative chain ） Superior sequencing . So the last insert DNA fragment It all comes from the first cDNA（ Negative chain ）, That is to say dUTP It's called fr-firststrand Why . In the process of sequencing, the positive chain is measured first reads, Then measure the negative chain reads（ Can distinguish positive and negative chains reads, This is the most fundamental difference from ordinary database building ）. In these reads When comparing to the reference genome , Those are compared to the genetic direction （ Justice chain direction ） Positive chain of reads Is the chain of justice reads, But those comparisons go in the opposite direction to the gene （ Antisense chain direction ） Positive chain of reads Is the antisense chain reads. So the same , Compare the negative chain in the direction of the gene reads Is the chain of justice reads, Compared to the opposite direction of the gene （ Antisense chain direction ） The negative chain of reads Is the antisense chain reads. So that eventually all justice chains reads And antisense chains reads Distinguish . So when determining gene expression levels , Can avoid gene antisense chain reads Matching interference , So as to more accurately detect the level of gene transcription and expression . and LncRNA The sequencing of DNA is also inseparable from the chain specific library building technology . There are three reasons. ：

1）lncRNA The source of is chain specific ;

2）lncRNA The source is the coding protein （mRNA）​ Antisense chains of genes , It is the natural antonym in the legend lncRNA（NAT-antisense lncRNA）; If it is a common non chain specific library , So the sequence is from mRNA, still NAT-antisence LncRNA It's hard to distinguish ;

3）​ Chain specific library construction can more accurately count the number of transcripts and determine the structure of genes , Accurately distinguish which chain of genome the obtained transcripts come from .​

Copyright notice ： This paper is about CSDN Blogger 「sixu_9days」 The original article of , follow CC 4.0 BY-SA Copyright agreement , For reprint, please attach the original source link and this statement .
Link to the original text ：https://blog.csdn.net/sixu_9days/article/details/81222407