2D Complete RNA

2D Complete RNA

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Library made using the 2D Complete RNA kit. Sample used was 10ng UHR post Ribodepletion. The library was sequenced on the Illumina 2500 Platform, 1x100 reads.

What would you do if you could simultaneously SEE EVERY RNA in your Sample?

Over 1300 unique miRNA and over 19,000 unique protein coding transcripts, along with all other RNA biotypes (including tRNA!) identified using single RNA-Seq library prep.

 


2D Complete RNA Libraries Provide Unparalleled Library Complexity, and rich data set even at ultra low inputs. 

 

Flat Alignment Stats across wide range of inputs


2D Complete RNA Libraries Provides Uniform Coverage Across the Gene Body of Transcripts

Templated used:  10ng Human Universal Reference RNA. Libraries were sequenced on an Illumina HiSeq2500 using single read mode (1x75 bp). This view of the 5´ to 3´coverage of RefSeq transcripts demonstrates consistent and uniform coverage for 2D Complete RNA libraries with 10ng. 


Far less PCR cycles required due to high conversion efficiency

About 3-5 cycles less than commonly used methods for similar inputs.

 


Even Coverage Across High GC Transcripts

Improved detection and uniform coverage obtained from a broad range of RNA concentration. Shown here is the DVL3 transcript, which contains regions of very high GC (outlined in black). GC rich regions are difficult to cover using other commercial kits.  The green traces represent different sample inputs: respectively from a top 0.1ng, 1ng, 10ng and 100ng of Human Brain mRNA. Bottom trace (Blue) was generated with 10ng of UHR RNA which was Ribo-depleted.

Uniform coverage obtained from a broad range of RNA concentration. Shown here is the coverage of GC-rich hemoglobin-alpha transcript (HBA1), which contains 62.8% GC bases, in Human brain mRNA at 0.1ng, 1ng, 10ug and 100ng (traces in order from top to bottom).

Uniform coverage obtained from a broad range of RNA concentration. Traces represent coverage of the FTL transcript, rich in GC content. Transcript is a difficult to cover using popular commercial kits. The green traces represent different sample inputs: respectively from a top 0.1ng, 1ng, 10ng and 100ng of Human Brain mRNA. Bottom trace (blue) was generated with 10ng of UHR RNA which was Ribo-depleted.


Excellent Coverage of Low Abundance Transcripts

Low-abundance transcript GLTPD1. Improved detection and uniform coverage obtained with broad range of RNA inputs using 2D Complete RNA kit. There is good coverage of GLTPD1 transcript which is otherwise difficult to cover using popular commercial kits.

The green traces represent different sample Human Brain mRNA inputs: respectively from top 0.1ng, 1ng, 10ng and 100ng of Human Brain mRNA. Bottom trace (blue) was generated with 10ng of UHR RNA which was Ribo-depleted.

Improved detection and uniform coverage obtained with broad range of RNA concentration of Low- abundance transcript MALAT1. Transcript is a difficult to cover using popular commercial kits.

The green traces represent different sample inputs: respectively from a top 0.1ng, 1ng, 10ng and 100ng of Human Brain mRNA. Bottom trace (blue) was generated with 10ng of UHR RNA which was Ribo-depleted.

Improved coverage of the whole transcript demonstrated on the housekeeping gene Beta-Actin. The back boxes highlights the improved coverage at the 3' and 5' regions

The green traces represent different sample inputs: respectively from a top 0.1ng, 1ng, 10ng and 100ng of Human Brain mRNA. Bottom trace (blue) was generated with 10ng of UHR RNA which was Ribo-depleted.


Reproducible data across A wide range of input - even in Picogram range

Data from libraries prepared using 2D Complete RNA kit with various input amounts of Human Brain mRNA. Data shows very consistent performance across 3 orders of magnitude of input range. Insets indicates Pearson coefficient of correlation. 


FFPE:  Robust performance on degraded & compromised sample types

Transcript coverage comparison between Liver FFPE vs Fresh/Frozen tissue.  

Libraries sequenced in Illumina HiSeq2500 (1x100bp read length).

Improved detection and uniform coverage obtained from a FFPE sample. Shown here is the DVL3 transcript, which contains regions of very high GC. GC rich regions are difficult to cover using other commercial kits.  The green traces represent RNA from Fresh-Frozen Normal Liver tissue and Blue plot represents RNA from FFPE Normal Liver tissue.

Low-abundance transcript GLTPD1. Improved detection and uniform coverage obtained with RNA from FFPE sample using 2D Complete RNA kit. There is good coverage of GLTPD1 transcript which is otherwise difficult to cover using other commercial kits.

The green traces represent RNA from Fresh-Frozen Normal Liver tissue and Blue plot represents RNA from FFPE Normal Liver tissue.

2D Complete provides precise and consistent measurement of RNA abundance. The high concordance between technical replicates of FFPE tissue indicates robust library prep performance and high reproducibility. Axes are log2(FPKM). R2 value is shown.


High reproducibility of 2D Complete RNA Kit is confirmed by ERCC analysis.

Reads mapping to ERCC data set from other experiments were pooled together and the FPKM was plotted against relative transcript abundance. All 92 ERCC transcripts were identified. 


 

2D Complete RNA: Molecular Biology & Mechanism of 'Continuous Synthesis'

The Mechanism of “Continuous Synthesis”:  2Dzyme adds the adaptor to cDNA using the mechanism of 'continues synthesis'.

During this process, an oligo dT primer (with partial adaptor) is annealed to the PolyA tail. 2Dzyme performs the reverse transcription reaction creating the cDNA strand till the 5' end of the RNA molecule using its strand-displacement and high-processivity properties. After reaching the 5’ end of the RNA template, 2Dzyme binds second adapter and continues synthesis without relying on the addition of non-templated bases or hybridization to the second adaptor.

The second adaptor is incorporated at high efficiency due to tight/preferential adapter binding by 2Dzyme. The result of this reaction is the cDNA product that includes template and both adapters sequence.  More than 99% of the library is automatically stranded due to 2Dzyme's directional reaction.  Adapters are engineered to prevent next (third) template binding.


Library Molecule Structure and Sequencing Recommendation:

Sequencing recommendation: 

- Compatible with Illumina Platform

- Dual barcode 

- We recommend Sequencing with Single-end reads. This can save on sequencing cost.

- The first 8 bases on Read 2 consist of a random tag sequence that can be used as an Unique Molecular Identifier (UMI). For complete Paired-end reads sequencing, please refer to the FAQ page.

- For Paired-end reads, there can be a significant drop in read quality for read 2 due to the presence of Poly T sequence.

- With 2D Complete RNA kit, single-read sequencing is sufficient to obtain coverage of full transcript.

- During analysis, 1st base should be trimmed before mapping, if it is not soft-clipped.