RNA seq stranded

Question

[ASid]

HI everybody. I am trying to understand a concept in RNA seq analysis if somebody could help. As far as i understand, in a stranded method (as in illumina true seq) first cDNA strand is sequenced in the dUTP method which is a reverse complement of sense strand/mRNA/coding strand. But I dont get one thing in it. If the first strand (which is a reverse complement of sense strand/mRNA/coding strand) is used in sequencing the reads, does this means that the reads in fastq files will be aligned to/or are complementary to sense strand in the gtf file? because I understand that the sequencers perform sequencing by synthesis and if we use the first strand of cDNA as template for sequencer we are going to get its complementary strand as a read output of sequencer?

[GenoMax]

You will find this collection useful.

[ASid]

Ok caggtaagtat. I got it. I have another question then. When we get one of the strands (with uracil which is complementary to mRNA) removed/digested. We have the other strand left. And when they say in sequencing protocol that we will amplify it and sequence it I understand that the amplification (clonal amplification in illumina) again would generate the complementary strand for the template strand that we are using then what is the use of digestion for the other strand?

Answer 1

[caggtaagtat]

Hi there,

the word "stranded" refers not to the coding direction of the gtf file, but wether the obtained reads are either complementary or equal to the sequence of the sequenced fragment.

In other words: first you have fragmented RNA which is used for the library preparation. During this preparation you can generate cDNA from the fragments. This cDNA is doublestranded during "unstranded library preparation protocols". This cDNA is then "sequenced" by synthesis, either from one strand or the other, generating two kinds of reads from one cDNA fragment of oposing directions.

The benefit of "stranded protocols" is that you manipulate cDNA generation, using for example the dUTP method, which enables you to break down one of the two cDNA strands right before actual sequencing, for example the second generated cDNA strand. Therefore you now only get one read sequence from an original fragment, which can be either the reverse complementary or the same sequence as the original fragment, depending on the used method.

See this graphic from bmc genomics:

[ASid]

Ok caggtaagtat. I got it. I have already read this paper but I still have the confusions. I have another question then. When we get one of the strands (with uracil which is complementary to mRNA) removed/digested. We have the other strand left. And when they say in sequencing protocol that we will amplify it and sequence it I understand that the amplification (clonal amplification in illumina) again would generate the complementary strand for the template strand that we are using then what is the use of digestion for the other strand?

[caggtaagtat]

So you mean, how the reverse stranded sequences are removed after cluster generation through amplification of a single sequence? So I'm only am familiar with Illumina a bit and I don't know exactly how it works, but there are two kinds of adapters at the ends of these amplified cDNA sequences, which allow the degradation of the sequences of the "wrong" direction. So at the end, theoretically, you have only cDNA sequences ín the clusters which have the same orientation (sense or antisense, depending on your protocol).