YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep

fast, flexible, and easy RNA-seq

 

YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Overview

RNA-Seq Services

Active Motif’s YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Kits provide a fast and easy method for either conventional Full-Transcript (FT) or 3’-Digital Gene Expression (DGE) sequencing.

Conventional full-transcript (FT) RNA-seq yields sequencing reads that map to the entire expressed transcriptome. The 3’ Digital Gene Expression (3’-DGE) option creates reads mapping just at the 3’-end of the transcript. Because of the reduced complexity, 3’-DGE requires a quarter to a third less sequencing depth, greatly reducing costs.

The YourSeq Library Prep workflow takes advantage of the natural phenomenon of DNA breathing to eliminate several steps which add time and cost to traditional RNA library prep protocols. DNA breathing is the opening and closing of DNA due to thermal fluctuations. Following first-strand cDNA synthesis, the YourSeq Library Prep adds the 5-prime adapter to the cDNA when the terminal end of the DNA:RNA hybrid molecule is open in a process called “Breath Capture.” This allows the enzymatic synthesis of a second strand of DNA without a step requiring the removal of RNA. This workflow also implements strictly enforced strand specificity, since the adapter is added by priming off the cDNA strand and cannot prime off the RNA strand.

YourSeq (FT & 3’DGE) Strand Specific Library Prep Kits can be ordered with either combinatorial dual indexes or unique dual indexes. Combinatorial dual-indexing (CDI) kits allow you to choose from 96 or 384 unique index combinations. Unique dual-indexing (UDI) kits allow the multiplexing of either 24 or 96 samples at a time without using any index twice. This can help avoid Illumina index hopping, or the incorrect assignment of libraries to the expected index.

YourSeq (FT & 3’DGE) Strand-Specific mRNA Library workflow(Click image to enlarge)

YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Highlights:

  • Only 4 hours from RNA to library prep
  • Option for both full-transcript (FT) or 3’-Digital Gene Expression (DGE) sequencing
  • Strictly enforced strand specificity
  • Available with Combinatorial Dual Indexing (CDI) or Unique Dual Indexing (UDI)

FT vs. 3’DGE

The YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Kits come with sufficient reagents for two types of RNAseq libraries, full-transcript (FT) or 3’-Digital Gene Expression (3’DGE). There are advantages and disadvantages to both methods. Use the appropriate workflow for your needs.

3’ Digital Gene Expression (3’DGE) Sequencing

The 3’ Digital Gene Expression (3’-DGE) option is optimized for differential gene expression analyses, where the main goal is to identify genes and pathways that are up- or down- regulated in response to some experimental condition.

Rather than use random priming, the cDNA synthesis step for 3’-DGE RNA-seq libraries uses an adapter with an oligo(dT) to prime off the transcript’s poly(A) tail. This means that 3’-DGE reads tend to map only to the 3’-end of the transcript. Since there is a theoretical 1-to-1 correlation between transcript molecule and cDNA molecule, transcript length is irrelevant and the number of reads mapping to a gene is solely a function of transcript abundance. A long gene, therefore, will have the same number of reads mapping to it as will a short gene of similar transcript abundance.

The reduced complexity associated with 3’-DGE confers one of the biggest advantages. Transcript counting means that less sequencing depth is needed for a robust analysis compared to conventional RNA-seq. Also, paired-end sequencing does not add value for 3’-DGE. So, a researcher who might sequence conventional RNA-seq libraries at 20-30 million pairs of paired-end reads per sample can reduce cost by sequencing those same samples as 3’-DGE libraries at 3-10 million single reads per sample.

3’ Digital Gene Expression (3’DGE) Sequencing

3’DGE RNA sequencing is optimal for:

  • Differential gene expression
  • Use with well-annotated genomes OR to improve annotation by identifying expressed genes and their 3’-UTRs
  • Genotyping and detection of polymorphisms in the 3’-UTR
  • Detection and characterization of alternative polyadenylation


Full Transcript (FT) Sequencing

The Full Transcript (FT) Sequencing option is for conventional full transcript RNA sequencing. This is the most common type of RNA sequencing that people are familiar with. Conventional full-transcript (FT) coverage RNA-seq yields sequencing reads that map to the entire expressed transcriptome. This is achieved through the combination of random RNA fragmentation and subsequent random priming during the cDNA synthesis step. The result is sequencing reads that, when mapped to the reference transcriptome or genome, tile across the exons of expressed transcripts. With conventional RNA-seq, the number of reads mapping to a gene is a function of transcript abundance and transcript length. A long gene, therefore, will have more reads mapping to it than a short gene of similar transcript abundance.

FT sequencing requires 20-30 million paired-end reads per sample, while 3’-DGE libraries require only 3-10 million single reads. However, there are circumstances where full-transcript sequencing is required. For example, splice variant analysis or the detection of exonic polymorphisms require reads from the entire coding sequence.

Full Transcript (FT) Sequencing

Full-transcript RNA sequencing is optimal for:

  • Genotyping and detection of exonic polymorphisms
  • Splice variant analysis
  • Detection of gene fusions abundance

YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Contents

All components should be stored at -20°C until first use. Once Carboxyl Beads have thawed, store at 4°C.

Catalog no. 23001 23002 23003 23004
Indexing CDI CDI UDI UDI
Reactions 24 96 24 96
Reagent  
FT Priming Mix 75 μl 300 μl 75 μl 300 μl
DGE Priming Mix 75 μl 300 μl 75 μl 300 μl
Control mRNA 5 μl 5 μl 5 μl 5 μl
First Strand Mix 135 μl 540 μl 135 μl 540 μl
RT enzyme 15 μl 60 μl 15 μl 60 μl
Breath Capture Mix 175 μl 690 μl 175 μl 690 μl
5-Prime Adapter 120 μl 480 μl 120 μl 480 μl
DNA Pol I 10 μl 30 μl 10 μl 30 μl
PCR Mix 264 μl 1056 μl 264 μl 1056 μl
Phusion Pol 8 μl 24 μl 8 μl 24 μl
Nuclease-Free Water 1.8 ml 1.8 ml 1.8 ml 1.8 ml
10 mM Tris HCl 1.8 ml 5 ml 1.8 ml 5 ml
Carboxyl Beads Buffer 1.8 ml 6 ml 1.8 ml 6 ml
Carboxyl Beads 1.5 ml 6 ml 1.5 ml 6 ml
YourSeq Index i5-A 30 μl 120 μl    
YourSeq Index i5-B 30 μl 120 μl    
YourSeq Index i5-C 30 μl 120 μl    
YourSeq Index i5-D 30 μl 120 μl    
YourSeq i7 1-24 Index Set 1 plate   1 plate  
YourSeq i5 1-24 Index Set     1 plate  
YourSeq i7 1-96 Index Set   1 plate   1 plate
YourSeq i5 1-96 Index Set       1 plate



 

YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep FAQs

What are the advantages of the YourSeq RNA-seq workflow?

The YourSeq Library Prep workflow takes advantage of the natural phenomenon of DNA breathing to eliminate several steps which add time and cost to traditional RNA library prep protocols. DNA breathing is the opening and closing of DNA due to thermal fluctuations. Following first-strand cDNA synthesis, the YourSeq Library Prep adds the 5-prime adapter to the cDNA when the terminal end of the DNA:RNA hybrid molecule is open in a process called “Breath Capture.” This allows the enzymatic synthesis of a second strand of DNA without a step requiring the removal of RNA. This workflow also implements strictly enforced strand specificity, since the adapter is added by priming off the cDNA strand and cannot prime off the RNA strand.

When do I choose Combinatorial Dual Indexing (CDI) versus Unique Dual Indexing (UDI)?

Combinatorial Dual Indexing (CDI) uses a unique combination of Index 1 (i7) and Index 2 (i5) indexes for each sample. However, samples may share index sequences. Unique Dual Indexing (UDI) uses a unique combination of Index 1 (i7) and Index 2 (i5) indexes for each sample, where no index is ever used more than once. Illumina sequencers using patterned flowcell technology like HiSeq, NovaSeq, and iSeq can show higher incidences of misassigned index assignment or index hopping. Using UDI can prevent such misassignments when using those instruments for sequencing.

What is RNA strand specificity?

mRNA transcripts can come from either the plus or minus strand of gDNA template. Strand-specific RNA library preps, enable you to determine the original orientation of the transcripts while non-stranded library protocols lose this information and you cannot tell whether a sequencing read came from the plus or minus strand of the gDNA template.

How does the kit ensure strand specificity?

With non-stranded RNA library prep, RNA transcripts are fragmented and random primers are used to create the first- and second-strand synthesis of cDNA before any adapters are added or fragments are amplified. As a result, the first and second cDNA strands of the two antisense transcripts look exactly the same and cannot be distinguished when sequenced. The YourSeq workflow adds an adapter to the first strand of cDNA only, while the transcript is still a cDNA-RNA hybrid, using a DNA dependent DNA polymerase (DNA Pol I) which primes off the cDNA strand and cannot prime off the RNA strand.

What is 3'DGE?

For 3’-DGE RNA-seq libraries, the cDNA synthesis step uses an adapter with an oligo(dT) to prime off the transcript’s poly(A) tail. This means that 3’-DGE reads tend to map only to the 3’-end of the transcript. Since there is a theoretical 1-to-1 correlation between transcript molecule and cDNA molecule, transcript length is irrelevant and the number of reads mapping to a gene is solely a function of transcript abundance. A long gene, therefore, will have the same number of reads mapping to it as will a short gene of similar transcript abundance.

What are the advantages of 3'DGE RNA-seq?

The reduced complexity associated with 3’-DGE confers one of the biggest advantages. Transcript counting means that less sequencing depth is needed for a robust analysis compared to conventional RNA-seq. Also, paired-end sequencing does not add value for 3’-DGE. So, a researcher who might sequence conventional RNA-seq libraries at 20-30 million pairs of paired-end reads per sample can reduce cost by sequencing those same samples as 3’-DGE libraries at 3-10 million single reads per sample.

What are the limitations of 3'DGE RNA-seq?

Because 3'DGE RNA-seq only covers the 3'end of the transcript, it's not appropriate for detection of exonic polymorphisms, splice variant analysis, or the detection of gene fusion abundance. That kind of information can only be determined by sequencing the whole transcript.

What are the advantages of Full Transcript RNA-seq?

Full Transcript RNA-seq is required whenever a determination can only be made from the whole RNA transcript. It should be used for the detection of exonic polymorphisms, splice variant analysis, or the detection of gene fusion abundance.

What are the limitations of Full Transcript RNA-seq?

The only drawback with Full Transcript RNA-seq is the increased cost incurred because at least 20-30 million paired end reads are required per sample, due to the increased complexity of the library.

What type of RNA input can be used?

The YourSeq (FT & 3'DGE) Strand-Specific mRNA Library Prep Kits can be used with either poly A enriched or ribosomal depleted mRNA.

What is rRNA depletion versus mRNA selection?

Ribosomal RNA (rRNA) depletion is a method that removes rRNA from total RNA, either during or after the isolation of total RNA so that the final result is enriched for mRNA. It can also be performed during library prep. Poly A enrichment uses oligo dT primers to selectively capture the polyadenylated 3' end of mRNA transcripts so that that the final result is enriched for mRNA.

How many sequencing reads are recommended?

For conventional full transcript (FT) RNA-seq libraries we recommend 20-30 million pairs of paired-end reads per sample. For 3’-DGE libraries we recommend 3-10 million single reads per sample.

Are there kit controls?

Each kit comes with drosophila melanogaster mRNA as a positive control.


 

YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Documents

 

 

 

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Name Format Cat No. Price  
YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Kit 24 CDI 24 rxns 23001 ¥6,570 Buy
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YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Kit 24 UDI 24 rxns 23003 ¥7,800 Buy
YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Kit 96 UDI 96 rxns 23004 ¥31,200 Buy