The SOLiD® SAGE™ Kit with Barcoding Adaptor Module provides a complete solution to perform digital gene expression profiling on the SOLiD® System. Instead of sequencing an entire transcriptome, the SOLiD® SAGE™ Kit generates 3’ expression tags—averaging 27 base pairs in length—enabling digital enumeration of gene expression profiles on the SOLiD® 4 System. Capturing only the 3’ end of transcripts allows for a cost-effective, highly sensitive elucidation of lowly expressed transcripts as SAGE™ tags lead to the representation of a majority of RefSeq genes (Figure 1 - click to enlarge). For an increased cost-effective sequencing solution, libraries from the SOLiD® SAGE™ Kit with Barcoding Adaptor Module can also be molecularly barcoded using the included barcoding adaptor module and a SOLiD® RNA Barcoding Module (sold separately).
SAGE™ Kit for SOLiD® Next-Generation Sequencing
High-throughput Digital Gene Expression Analysis
- SOLiD® SAGE™ Kit with Barcoding Adaptor Module
- Limited data for analysis required by focusing on 3’ transcripts
- Greater mappability using the included SOLiD® SAGE™ software package
- Compatible with SOLiD® RNA Barcodes for multiplexing capabilities
Digital profiling using the SOLiD® SAGE™ System
SOLiD® SAGE™ 27 bp tag library generation
The SOLiD® SAGE™ Kit has been optimized to construct libraries with SAGE™ tags and SOLiD® specific adapters for pre-amplification and/or emulsion PCR directly. The kit includes reagents that generate longer tags (27 bp vs. 21 bp) compared to previous SAGE™ methods. In addition, incorporating the recommended protocol improvements allow for expression profiling with and without large scale PCR amplification (Figure 2).
After sequencing, the tags are mapped to a reference sequence database and differential expression is determined by counting the number of tags representing a digital enumeration of each unique transcript in the original sample.
Sensitive gene expression using the SOLiD® SAGE™ Kit
Evaluation of expression differences in reference RNAs HBRR and UHRR was performed using real-time RT-PCR, bead microarrays, and the SOLiD® SAGE™ Kit on a subset of 25 diagnostic genes. Higher correlation was observed between real-time RT-PCR and the SOLiD® SAGE™ Kit (R = 0.956) than between the real-time RT-PCR and microarray platform (R = 0.916) (Figure 3A, 3B), validating the accuracy of expression profiles from the SOLiD® SAGE™ Kit. Furthermore, the SOLiD® SAGE™ Kit demonstrated a higher pearson correlation at low expression levels.
Massively parallel sequencing using the SOLiD® 4 System yields 2–4 million RefSeq-mapped tags per SOLiD® SAGE™ Kit reaction on 1/8 of a sequencing slide. The system generates data that is highly reproducible and correlates very well with real-time RT-PCR results from the same sample. The percentage mapped using the new SAGE™ Software v1.10 to RefSeq (with genomic mapping capability) on the SOLiD® 4 System is on average four-fold greater than samples analyzed with the previous v1.06 SAGE™ mapping method (Figure 4).
Figure 4: Comparative library mapping using the new SAGE™ Software v1.10 to RefSeq (with genomic mapping capability) on the SOLiD® 4 System to the previous v1.06 SAGE™ mapping method. Eight replicate libraries were generated and barcoded [BC1–BC8] from Ambion® brain HBRR total RNA . Eight replicate libraries were also generated and barcoded [BC9–BC16] from Universal total RNA (Stratagene). The sixteen samples were then pooled prior to emulsion PCR and were run on a single slide on the SOLiD® 4 System. The samples were mapped to RefSeq using the old v1.06 software method, which uses the 27_0 parameter and on the new v1.10 software method using the 22_1 mapping parameter.