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Utilize the tremendous sequencing capacity of SOLiD® System with library multiplexing.

Reach Your RNA sequencing Goals with Maximum Efficiency

  • Multiplex whole transcriptome, small-RNA, and SAGE™ libraries
  • Unique barcode sequences provide optimal multiplexing
  • Three kits available

Barcoding in RNA applications is advantageous when examining differentially expressed genes within a time course series or between drug treatments. SOLiD® RNA Barcoding Kits conveniently multiplex whole transcriptome and small RNA libraries constructed by the SOLiD® Total RNA-Seq Kit, and libraries constructed by the SOLiD® SAGE™ Kit.

Using unique barcode sequences for optimal multiplexing of up to 48 libraries, SOLiD® RNA Barcoding Kits enable the assignment of a unique identifier to templated beads made from a single library. Once the identifiers are assigned, multiple batches of templated beads may be pooled together for emulsion PCR and then sequenced.

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Figure 1. Barcoded RNA library design.

Three kits are available. Each contains 16 different barcodes selected for

  • Uniform melting temperature
  • Low error rate
  • Orthogonal sequences unique in color space

Barcodes are added to the target sequence 3' end using a modified P2 adapter (figure 1).  SOLiD® RNA Barcoding Kits are compatible with SOLiD® Total RNA-Seq and SOLiD® SAGE™ protocols, making it easy to integrate barcodes during library construction (figure 2).

Figure 2. The SOLiD® RNA Barcoding Workflow. Barcoded RNA libraries are generated at the cDNA amplification step in the RNA library preparation procedures. Each barcoded library is handled separately through purification and completion of the library preparation. Barcoded libraries are ready to be combined into a multiplex sequencing pool just prior to templated bead preparation by emulsion PCR.

Following sequencing of the target, additional rounds of ligation-based sequencing are performed using primer sets complimentary to the barcode. The resulting reads are sorted by the barcode and aligned in groups to the reference sequence, enabling sequencing of multiple libraries on the same slide—maximizing throughput while dramatically reducing costs.

Figure 3. Click to enlarge. High Mapping Accuracy with Uniform Representation Across SOLiD® RNA Barcodes 1-96. 96 barcoded whole transcriptome libraries were generated from HeLa poly(A) mRNA using the SOLiD® Total RNA-Seq Kit and SOLiD® RNA Barcoding Kits, Modules 1-16, 17-32, & 33-48, 49-64, 65-80, & 81-96. The Applied Biosystems WTv1.2 pipeline was used to map reads against the Human genome (hg18) and a filter database consisting of repetitive elements and adaptor sequences. The stacked barplot above shows the percentage of total mappable reads for each of the 96 libraries that properly map to the genome between 1 and 10 times, map too many times (>10), map to the filter database or do not map at all. Over 350 million mappable reads were generated in total with the SOLiD® 4 System. Properly mapped reads range from 65%-75% across all 96 barcodes. This not only represents highly accurate mapping, but also suggests very similar performance of each barcode across the differ­ent libraries. The presence of filtered reads, primarily representing libraries without inserts, ranges from ~2%-15%; reflecting the low level of variability associated with the SOLiD® Total RNA-Seq method.