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Old 11-28-2011, 02:57 AM   #1
papori
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Default strand-specific cDNA

i know that the question is stupid, but i am not sure that i am understanding it.
i dont see the problem of recognize the sense RNA from double strand cDNA.
isnt it the one with polyA?
or,
i dont understand what is strand-specific cDNA ?

thanks
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Old 11-28-2011, 04:04 AM   #2
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Sure, but many of the sequence reads will not have the polyA part in them. So one is not certain whether they derive from sense transcripts, or anti-sense.

That said, I don't know that strand specificity is essential for de novo transcriptome derivation. Helpful, perhaps, but not essential.

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Old 11-28-2011, 04:43 AM   #3
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ok, but..
why many of the sequence reads will not have the polyA part in them?
all mRNA molecule have polyA, that is how we can make cDNA.
we reverse transcrpitase by polyT.
so when is the level that part of the mRNA/cDNA loosing their polyA tail?

thanks again..
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Old 11-28-2011, 04:52 AM   #4
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The vast majority of RNAseq protocols use fragmented RNA or cDNA for library construction. This is because there are limits on the length of PCR products that can be generated in a PCR reaction. Or rather, I should write, short PCR products are more robustly amplified than long ones.

Also, even if you see that polyA tail, you are still inferring that is the transcribed strand. Whereas you can use strand-specific methods to assay which strand it is. Get it? One is a presumption you are making (whether for good reason or not), the other is an observation.

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Old 11-28-2011, 05:18 AM   #5
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ok, thank you phillip, i think i got it.

so if i undersood tou right.
when converting mRNA to cDNA, i can get the full length of the mRNA as cDNA.
but after, when i want to create library(probably for PCR amplification) i have to break the cDNA because DNA polymerase has limit around 3K bases.

so, when i want to amplificate the library that i just broke to fragments, i must use random primers and not just polyT?

thanks
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Old 11-28-2011, 06:21 AM   #6
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Okay, we are starting to get into the details of RNAseq here. But most commonly RNAseq library protocols follow these steps:

(1) Ribodepletion (removing most of the ribosomal RNA that composes the majority of all total RNA preps.)
(2) RNA fragmentation. For Illumina this fragmentation will generate a peak around a few hundred nucleotides.
(3) First and second strand cDNA synthesis. Because of the RNA fragmentation step, the majority of cDNAs will be in the target range.
(4) ds cDNA end polishing, adapter ligation

After you ligate the adapters to the cDNA, then you have your library of amplicons. These are usually subjected to other manipulations, but eventually end up being diluted to low titres and amplified by cluster or emulsion PCR to create ~ 1um splotches of area on a flow cell surface with thousands or millions of copies of the same amplicon. Sequencing proceeds from a priming site in one of the adapters.

Why a few hundred bases instead of something close to the polymerase size limit?

First, your read length is only 50-100 nt or so. Reading from the very ends of full length cDNAs will only interrogate a percentage of your entire transcriptome. To read the entirety you either need very long read lengths, or you need to fragment your transcripts prior to sequencing them. Second, PCR is much more efficient at replicating smaller amplicons than larger ones. So by going shorter you get a signal boost for your template clusters or beads.

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Old 11-29-2011, 11:07 PM   #7
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thank you Phillip!
that was helpful!
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Old 11-30-2011, 12:10 AM   #8
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Does any one know the difference between T7 RNA polymerase based ivt to dUTP second strand synthesis?

thanks
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Old 11-30-2011, 02:40 AM   #9
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Quote:
Originally Posted by papori View Post
Does any one know the difference between T7 RNA polymerase based ivt to dUTP second strand synthesis?

thanks
T7 IVT is an linear amplifcation step. Basically, double stranded cDNA is created using an oligo dT that also includes the T7 promoter sequence. T7 polymerase is then used to generate cRNA as it only transcribes from DNA downstream of the T7 sequence. It generates amplified strand specific cRNA.
Typically it's used for expression microarrays that require large amounts of material for hybridisiation.

The dUTP RNA seq method uses an oligo dT to prime mRNA, then deoxyuridine triphosphate instead of deoxythymidine triphosphate is used in the synthesis of the second strand of cDNA. cDNA is then fragmented and a library prepared (fragmentation, end repair and ligation). Before the final PCR amplification step, an enzyme (Uracil-N-Glycosylase) is used to cut the second strand at all the uracil bases, so only the first strand can be sequenced - maintaining the strand specificity.
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Old 11-30-2011, 04:49 AM   #10
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Quote:
Originally Posted by TonyBrooks View Post
T7 IVT is an linear amplifcation step. Basically, double stranded cDNA is created using an oligo dT that also includes the T7 promoter sequence. T7 polymerase is then used to generate cRNA as it only transcribes from DNA downstream of the T7 sequence. It generates amplified strand specific cRNA.
Typically it's used for expression microarrays that require large amounts of material for hybridisiation.
Though not an exponential amplification, T7 RNA polymerase does produce quite a few copies of RNA from each DNA strand (by displacement of the nascent strands, I presume).

Affy had a protocol (the 2 cycle amplification method) that converted the RNA produced by T7 back into cDNA followed by another cycle of T7 amplification. That gave near-exponential amplification.

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Old 12-01-2011, 12:55 AM   #11
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I joined that thread because the first post was a "stupid" question and those are often the most interesting ones.. Thanks Phillip and Tony for the details and explanations.
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Old 12-01-2011, 01:12 AM   #12
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Thanks TonyBrooks & phillip
this is great explanation!
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Old 12-01-2011, 06:53 AM   #13
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what is the reason to the fact that when we are talking about "strand specific" dUTP is better than 7T IVT?
only the linear vs. exponential amplification?
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Old 12-01-2011, 07:21 AM   #14
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T7 IVT is not a library prep method. It is a way of amplifying poly A+ RNA. It produces single stranded cRNA (although it does keep it's strandedness as it's only amplified from one strand). It's mostly used in microarrays where the input needs to be single stranded material.

The "dUTP" method is a library prep method. The only amplification is at the end of the protocol when the sample is enriched for adapter ligated material by PCR. Before this step, the second strand (marked with uracil) is degraded to only the first strand is enriched by PCR.

Amplification is a red herring here. You can't really compare dUTP and T7 IVT as they are predominantly used for different technologies. Although both methods retain strandedness, it's not an issue with T7 IVT as the microarray will only have probes where the strand of the mRNA is known. Strandedness is much more important in sequencing as you want to know which strand a read has come from to identify novel transcripts.
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Old 12-01-2011, 08:57 AM   #15
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Maybe i just didnt understand what i read from this paper:
Development and applications of single-cell transcriptome analysis
Fuchou Tang1,2, Kaiqin Lao3 & M Azim Surani1

Combined with the existing strand-specific cDNA library preparation strategies, such as T7 RNA polymerase–based in vitro transcription and dUTP second-strand synthesis strategies it would be possible solve the stranded-ness of mRNAs is lost in the library construction, which prevents discrimination between sense and antisense transcripts from the same locus.

isnt it said that combined with both will solve the stranded-ness of mRNAs??
Can you explain that?

Thanks,
pap
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Old 12-02-2011, 06:51 AM   #16
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Quote:
Originally Posted by papori View Post
Maybe i just didnt understand what i read from this paper:
Development and applications of single-cell transcriptome analysis
Fuchou Tang1,2, Kaiqin Lao3 & M Azim Surani1

Combined with the existing strand-specific cDNA library preparation strategies, such as T7 RNA polymerase–based in vitro transcription and dUTP second-strand synthesis strategies it would be possible solve the stranded-ness of mRNAs is lost in the library construction, which prevents discrimination between sense and antisense transcripts from the same locus.

isnt it said that combined with both will solve the stranded-ness of mRNAs??
Can you explain that?

Thanks,
pap
OK. I've just read the paper. I think it's a bit confusing here as the paper is predominantly about single cell transcription profiling. In order to do this, you will need to amplify your starting material as there's no way you'll get enough library yield from one cell without amplification.

In fact, what the paper says is:

"Combined with the existing strand-specific cDNA library preparation strategies, such as T7 RNA polymerase–based in vitro transcription and dUTP second-strand synthesis strategies, it will be possible to recover the strandedness information for single-cell transcriptomes in the near future"

As I understand, it is implying that you could use T7-IVT to amplify into cRNA then from that create a cDNA library, with uracil incorporated into the second strand. This can then go into library prep, have the second strand degraded and finally PCR amplifed. It might give you enough material so to sequence whilst maintaining strandedness. Although IVT is a very 3' biased amplification, so there are probably going to be issues with this approach.
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Old 12-03-2011, 09:05 AM   #17
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so if i understood you well, T7-IVT is only the amplification step because the low amount of RNA in single cell. after use T7-IVT, we will degraded the cRNA.
then - we will use the dUTP method to create cRNA for the PCR step?

why do we have to use T7-IVT for the first step?(it not so clear...)
Double amplification?
maybe because T7-IVT have less by-products?

Thanks TonyBrooks!
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Old 12-04-2011, 07:41 AM   #18
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Quote:
Originally Posted by papori View Post
so if i understood you well, T7-IVT is only the amplification step because the low amount of RNA in single cell. after use T7-IVT, we will degraded the cRNA.
then - we will use the dUTP method to create cRNA for the PCR step?

why do we have to use T7-IVT for the first step?(it not so clear...)
Double amplification?
maybe because T7-IVT have less by-products?
Yes, you get higher levels of amplification. The T7 step can effectively bump the amount RNA up 100-fold or so -- at the cost of introducing high 3' bias (and probably also other biases.)

I would avoid it, where possible. I think the main reason it is still used is that the earlier, microarray, techniques frequently made use of it.

Actually it isn't really the T7 RNA polymerase that is producing the 3' bias, but the oligo dT primer used for first strand synthesis. It is just one of those quick, sloppy methods that "solve" 2 problems at once (1) avoiding most of the signal from ribosomal RNA and (2) amplifying the starting material.


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Old 11-26-2013, 05:57 PM   #19
chaomeizhang
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Question How Final PCR amplification of first stand cDNA only amplify first strand cDNA

Quote:
Originally Posted by TonyBrooks View Post
T7 IVT is an linear amplifcation step. Basically, double stranded cDNA is created using an oligo dT that also includes the T7 promoter sequence. T7 polymerase is then used to generate cRNA as it only transcribes from DNA downstream of the T7 sequence. It generates amplified strand specific cRNA.
Typically it's used for expression microarrays that require large amounts of material for hybridisiation.

The dUTP RNA seq method uses an oligo dT to prime mRNA, then deoxyuridine triphosphate instead of deoxythymidine triphosphate is used in the synthesis of the second strand of cDNA. cDNA is then fragmented and a library prepared (fragmentation, end repair and ligation). Before the final PCR amplification step, an enzyme (Uracil-N-Glycosylase) is used to cut the second strand at all the uracil bases, so only the first strand can be sequenced - maintaining the strand specificity.
I read many posts about dUTP strand specific RNA-seq, nobody explained why final PCR amplification step after degradation of second strand of cDNA only amplify the first strand of cDNA. My understanding is PCR always generates double stranded amplicoms. Can anybody answer the question. Thanks in advance.
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Old 11-26-2013, 11:10 PM   #20
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It is because you have two different adaptors (annealed in a Y-shape). Without dUTP you will get two different amplicons for each insert due to the Y-adaptors meaning that the first read can start from either end of the insert, but with dUTP you will only amplify one amplicon (which will be double stranded) so all reads from the same transcript will go in the same direction.
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