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Feb 15, 2011 (Vol. 31, No. 4)

Reshaping Whole Transcriptome Profiling

RNA-Seq Sample-Prep Reagents Aim to Help Analyze Previously Inaccessible Samples

  • Profiling the HIV Genome

    Click Image To Enlarge +
    Figure 2. Differential expression and alternative splicing of tropomyosin 2 (TPM2). Data from UCSC genome browser illustrating read coverage of a portion of the TPM2 gene in human colon tumor vs. normal adjacent (NAT) samples. The data shows a concordant several-fold reduction in the TPM2 transcript abundance in tumor in both FFPE and fresh frozen samples (note the different Y-axis scales). An alternative splicing event at Exon6 of the TPM2 gene is also seen in both the fresh frozen and FFPE samples (black arrows).

    It is estimated that 38 million people worldwide are infected with human immunodeficiency virus (HIV), with an additional 4.1 million people infected each year. With the growing HIV+ population it is important to understand how this virus mutates and develops drug resistance over time, which provides additional challenges in developing effective therapies.

    Researchers at the University of California, Berkeley lead by Stephanie Willerth chose RNA-Seq to study HIV evolution in clinical isolates through a detailed analysis of viral sequences. However, there are several challenges in utilizing NGS to perform such studies. In particular, the amount of HIV RNA that can be obtained from clinical blood samples is low and does not provide enough material for traditional library construction protocols.

    PCR amplification could not be used as PCR can introduce bias into a sample since only sequences that contain significant homology with the primers sequences will be amplified, which in turn affects the resulting analysis of diversity present in the HIV genomes.

    In order to obtain sufficient quantities of material for NGS analysis, the researchers utilized the Ovation RNA-Seq System for low bias amplification of HIV genome samples to produce double-stranded cDNA, which was followed by library construction and sequencing on the Illumina Genome Analyzer platform.

    The results produced high genome coverage using 36 base pair reads enabling accurate comparison of sequence differences. The researchers are currently developing metrics for evaluating HIV diversity and evolution and how these parameters change in response to long-term antiviral drug therapy. The methods developed in this study for low input RNA amplification can also be applied to other clinically relevant viruses.

    FFPE tissue fixation has been the clinical sample archival storage method of choice for many years, in particular for cancer research and diagnostics. FFPE samples can be connected to patient data through healthcare, disease, and population registries and represent an important resource for retrospective studies of genomic-based disease.

    While this preservation method renders the samples suitable for traditional cell and tissue characterization by microscopy, the FFPE samples are typically difficult to access for genomic analyses. RNA and DNA exposed to formalin fixation are often degraded and cross-linked, thereby making these samples refractory to the steps used in NGS analysis.

    In order to access this important category of samples, NuGEN has developed the Ovation RNA-Seq FFPE System which is optimized for degraded RNA typically found in FFPE samples. Figure 2 shows an example of differential expression for a specific gene (TPM2) detected in a human colon tumor compared to normal adjacent tissue from FFPE and fresh frozen (FF) samples. The data show concordant several-fold reduction in the TPM2 transcript abundance in tumor in both FFPE and FF samples as indicated by the read distribution across exons. In addition, an alternative splicing event at Exon6 of the TPM2 gene is also seen in both the fresh frozen and FFPE samples (black arrows).

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