Total Solution for RNA-Cap Sequencing

Background

High throughput sequencing has been widely used in clinical research due to its time-saving and low-cost in genomic analysis. Unlike DNA-seq, the RNA-seq can associate gene expression with biological events and cell states. The gene expression is coefficient with genetic and epigenetic information, such as methylation and histone modification. However, the sensitivity is always limited by the uneven length and abundance of transcriptome.

The RNA targeted capture can enrich interested RNA regions, which can significantly improve the coverage and sensitivity of sequencing, and detect low-frequency transcripts with high efficiency. Moreover, the RNA-Cap can also be flexibly customized to detect hundreds of gene regions at a time.On the basis of that,Nanodigmbio Technology provides a total solution for RNA-Cap sequencing on both MGI platforms and Illumina® platforms.

Solution

RNA-Cap solution 1

RNA-Cap solution 2

cDNA Synthesis

Library Preparation

Blocking

Target Capture

NadPrep Total RNA-To-DNA Module

NadPrep DNA Universal Library Preparation Kit (for MGI)

NadPrep NanoBlockers (for MGI)

Custom Panel

NadPrep DNA Universal Library Preparation Kit (for Illumina®)

NadPrep NanoBlockers (for Illumina®)

NadPrep Hybrid Capture Reagents

For research use only. Not for use in diagnostic procedures.

dsDNA yield from different amount of RNA input

Figure 1. dsDNA yield from different input amount of cell RNA (10 ng, 50 ng, 100 ng and 200 ng) using NadPrep Total RNA-To-DNA Module.

High Efficiency of Target Enrichment

Figure 2. Performance of Total RNA-Seq，rRNA-depleted RNA-Seq and RNAcap-Seq. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with NadPrep DNA Universal Library Preparation kit (for MGI). NanOnco Plus Panel v2.0 was used for RNAcap-Seq. A. RNAcap-Seq showed the lowest rRNA proportion in both K562 cell lines and FFPE tissues. B. RNAcap-Seq showed the highest enrichment efficiency for exon regions. C. RPKM values (in Reads per Kilobase of Exon per Million Reads) differ RNA-seq methods for libraries prepared from FFPE tissues. RNAcap-Seq can significantly enrich the targeted area for 20-50 times.

High Performance of Single Capture

Figure 3. Comparison of single capture and double capture with different panels on various NGS platforms. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with either NadPrep DNA Universal Library Preparation kit (for MGI) or NadPrep DNA Universal Library Preparation kit (for Illumina®), and were captured by different panels. A-B. Sequencing results on MGI platform (MGISEQ-2000, PE100); C-D. Sequencing results on Illumina® platform (Novaseq 6000, PE150). The results indicate that single capture can achieve an efficient enrichment similar to double capture.

Compatible with Multiple Sample Types

Figure 4. The effect of FFPE sample quality on RNA-Cap. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with NadPrep DNA Universal Library Preparation kit (for MGI), and were captured by NanOnco PlusPanel . A. The correlation between rRNA proportion and DV200. B. The correlation between on-target rate and DV200. For high quality FFPE samples (DV 200 > 60%), the rRNA ratio <10% and on-target rate > 80%; For highly degraded FFPE samples (DV 200 <30%), the rRNA ratio significantly increased with the capture performance declined. The DV 200 reveals the proportion of RNA fragments over 200 nucleotides.

Assessment of gDNA Contamination

Figure 5. Assessment of gDNA contamination in RNA samples. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with NadPrep DNA Universal Library Preparation kit (for MGI), and were captured by NanOnco Plus Panel v2.0. The specific targeted regions (known as the non-coding regions) are recommended to evaluate the DNA contamination. The threshold of contamination for RNA-Cap is required (10 read counts/Gb data, for instance).

Gene Fusion Detection

Figure 6. Fusion detection of FFPE RNA standard. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with NadPrep DNA Universal Library Preparation kit (for MGI), and captured by NanOnco Plus Panel v2.0. The test samples are gradient-diluted Onco Fusion FFPE RNA Reference Standard (GeneWell, GW-OPSM001), and the ordinate is the normalized Spanning Reads.

Compatible with MGI Platform and Illumina® Platform

Figure 7. Comparison of quality control parameters of RNA-Cap sequencing on various platforms. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with either NadPrep DNA Universal Library Preparation kit (for MGI) or NadPrep DNA Universal Library Preparation kit (for Illumina®), and were captured by NanOnco Plus Panel v2.0 combined with Ext-RNA Control Panel. The quality control parameters of RNA-Cap sequencing on MGI platform (MGISEQ-2000, PE100) and Illumina® platform (Novaseq 6000, PE150) with RNA from K562 cell lines and Onco Fusion FFPE RNA Reference Standard (GeneWell, GW-OPSM001) reveal no significant difference, including A. on-target ratio, B. rRNA ratio, C. ERCC ratio and D. ERCC count.

Figure 8. Transcript coverage of RNA-Cap sequencing on various platforms. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with either NadPrep DNA Universal Library Preparation kit (for MGI) or NadPrep DNA Universal Library Preparation kit (for Illumina®), and were captured by NanOnco Plus Panel v2.0 combined with Ext-RNA Control Panel. The transcript coverage of RNA-Cap sequencing on MGI platform (MGISEQ-2000, PE100) and Illumina® platform (Novaseq 6000, PE150) with RNA from A. K562 cell lines and B. Onco Fusion FFPE RNA Reference Standard (GeneWell, GW-OPSM001) reveal uniformity.

Figure 9. Comparison of RPKM on various platforms. Libraries were prepared using NadPrep Total RNA-To-DNA Module coupled with either NadPrep DNA Universal Library Preparation kit (for MGI) or NadPrep DNA Universal Library Preparation kit (for Illumina®), and were captured by NanOnco Plus Panel v2.0 combined with Ext-RNA Control Panel. The RPKM of RNA-Cap sequencing on MGI platform (MGISEQ-2000, PE100) and Illumina® platform (Novaseq 6000, PE150) with RNA from A. K562 cell lines and B. Onco Fusion FFPE RNA Reference Standard (GeneWell, GW-OPSM001) was compared.