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Whole Transcriptome Amplification Sequencing Technology (SEQR)

The SeqPlex RNA Amplification Kit for whole genome transcriptome amplification (WTA) is designed to facilitate next-generation sequencing (NGS) of extremely small quantities of degraded/highly fragmented RNA including non-polyA-tailed RNA. The yields from serum, single cells, RNA immunoprecipitation (RIP) or formalin-fixed paraffin-embedded tissue samples (FFPE) are often less than required for successful NGS library preparation. The SeqPlex kit allows the user to pre-amplify these RNA samples while maintaining patterns of differential expression found in the unamplified sample, prior to entering an NGS workflow.

This kit is an extension of the WTA product line and has been developed to integrate with the Illumina® (GAIIx sequencer), SOLiD™ System (from Life Technologies), Ion Torrent (from Life Technologies) and other next generation sequencing workflows.

Features and Benefits

  • Amplify fragmented or intact RNA from all sources including FFPE and RIP
  • Microgram quantities of double-stranded cDNA from picogram quantities of total RNA in approximately 8 hours
  • Semi-degenerate + oligo dT library primer mix for more complete uniform amplification and transcriptome coverage and efficient priming
  • No need to fragment DNA before sequencing
  • Compatible with next generation sequencing workflow platforms (except Pacific Biosciences and Helicos systems)

WTA Procedure

WTA Procedure

SeqPlex RNA Amplification

The SeqPlex RNA Amplification kit was used to amplify 2.5ng RNA from Ambion's Human Brain Reference Total RNA (Brain) and Stratagene's Universal Human Reference RNA (UHR). Deep sequencing was performed with both unamplified and SeqPlex-amplified RNA on an Illumina HiSeq2000. Approximately 84% of reads from SeqPlex-amplified samples align with the human genomic reference compared with 96-97% for unamplified. Results show > 90% of the primer sequences are removed by the SeqPlex primer-removal procedure for both amplified samples. The residual 9% primer sequence accounts for most of the non-aligned reads.

Overlap between genes detected for unamplified and SeqPlex-amplified RNA

Overlap between genes detected for unamplified and SeqPlex-amplified RNA. Approximately 90% of the aligned reads from SeqPlex-amplified aligned to the same Refseq genes as for unamplified.

SeqPlex library synthesis uses semi-degenerate priming for RNA amplification

SeqPlex library synthesis uses semi-degenerate priming for RNA amplification without 3’bias. Refseq transcripts' position (per cent distance from the 5’ end) mapped against the number of reads at that position (above) shows similarly uniform coverage for unamplified and SeqPlex-amplified total RNA.

SeqPlex RNA Amplification kit maintains representative transcript levels during amplification

SeqPlex RNA Amplification kit maintains representative transcript levels during amplification. The plot below of amplified versus unamplified differential expression (human brain/UHR) shows a correlation coefficient approaching 0.9, confirming the capability of the SeqPlex RNA amplification kit to perform with minimal bias.

Ordering Information

Product No. Description Quantity Add to Cart
SEQR SeqPlex RNA Amplification Kit SEQR-10Rxn
SEQR SeqPlex RNA Amplification Kit SEQR-50Rxn
SEQR SeqPlex RNA Amplification Kit SEQR-500Rxn Call customer/technical service to inquire