How Do We Count Transcripts?
To measure transcriptome variation, we first isolate RNA from the tissue(s)
of interest, then convert the RNA to DNA for sequencing. Multiple samples
can be tagged with molecular barcodes to increase sample throughput
(as shown in Fig 1).
Figure 1
DNA pools are sequenced by massively parallel sequencing
using the Illumina Genome Analyzer (Fig 2).
Figure 2
This technology reads a small
portion, or microread, of every DNA in the sample. Millions of microreads are
recorded per sample, and these microreads are mapped back to a genome or
transcriptome reference to identify which genes are expressed in RNA pool
(Fig 3).
Figure 3
Under different experimental conditions, genes may be constantly
expressed (as indicated by black microreads that map to two genes), or they
may be differentially expressed as shown with red and blue microreads
(gene 1 is only expressed in the red treatment). When properly conducted,
MPS can be used to discover the sequence of expressed genes and their abundance.
Transcriptomes Evaluated to Date
To date, our collaboration has sequenced a staggering 42 billion bases of DNA from 128 different transcriptome and genome sources. This amount of sequence information dwarfs the human genome, which contains about 3.3 billion bases of DNA.
Transcriptome sequencing by region. PNW, Pacific Northwest Research Station; PSW, Pacific Southwest Research Station; RMRS, Rocky Mountain Research Station.
Data Deposition
Once checked for accuracy, all sequencing data will be deposited to the NCBI Short Read Archive.
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Analysis of complete chloroplast genomes (120,000 bp) identifies sites unique to pine species. |
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