• Abstract


• Methods


• Data


• Results


• Programs

ABSTRACT

 
RACE (Rapid Amplification of cDNA Ends) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. Here, we describe a strategy that uses array hybridization to improve sampling efficiency of human transcripts. The products of the RACE reaction are hybridized onto tiling arrays, and the exons detected are used to delineate a series of RT-PCR reactions, through which the original RACE mixture is segregated into simpler RT-PCR reactions. These are independently cloned, and randomly selected clones are sequenced. This approach is superior to direct cloning and sequencing of RACE products: it specifically targets novel transcripts, and often results in overall normalization of transcript abundances. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of novel transcripts, and we investigate multiplexing it by pooling RACE reactions from multiple interrogated loci prior to hybridization.

METHODS

• Simulation of RACEarray transcript sampling

DATA

• Experimental verification


• Genes


• RACEfrags


• Pairs of RT-PCR primers (README)


• Results of RT-PCR and cloning and sequencing:


• "Raw" sequencing reads (contigs + singlets, low quality and vector sequences trimmed out. Includes positive controls) (FASTA format)


• BLAT mapping of sequencing reads, followed by alignment-based clustering of redundant sequences (PSL format)


• Multiplexing of RACEarray normalization


• Optimal number and combination of tissues


• Index genes


• Index exons


• Primers


• Initial RACEfrags


• Assigned filtered RACEfrags


• Nomenclature for the raw RACEfrag files


• Complementary information about the index genes


• Optimal distribution of exons to RACE per locus


• Index genes


• Index exons


• Primers


• Initial RACEfrags


• Assigned filtered RACEfrags


• Nomenclature for the raw RACEfrag files


• Complementary information about the index genes


• Pooling of RACE reactions - Genomic extent of loci


• Index genes


• Index exons


• Primers


• Initial RACEfrags


• Assigned filtered RACEfrags


• Nomenclature for the raw RACEfrag files


• Complementary information about the index genes

RESULTS

• RACEarray discovery of transcript isoforms
  
  Following is a more detailed version of figure 2b from the main text (i.e. with Ensembl annotations and fully expanded EST and mRNA tracks).
  We also provide two extra detailed figures representing RT-PCR verification of novel RACEfrags in different index loci.
  See main text for legend.


• Figure 2b (CHAF1B locus)


• KCNJ15 locus

RT-PCR-verified connectivity between an intergenic 5' RACEfrag and its index locus, KCNJ15 (a.k.a. NM_170736).
Ten novel sequences were discovered, of which seven connect the two RT-PCR primers used.


• SFI1 locus

RT-PCR-confirmed chimeric transcript.
A 5' RACEfrag, lying within an exon of DRG1, connects with its index exon in locus RP5-858B16.1, 80 kilobases away, on the same strand.
Note the intervening locus (RP11-247I13.2) on the opposite strand.

• Multiplexing of RACEarray normalization


• Optimal number and combination of tissues: Characteristics of Q1 RACEfrags


• Optimal distribution of exons to RACE per locus: Characteristics of Q2 RACEfrags


• Pooling of RACE reactions -- genomic extent of loci: Characteristics of Q3 RACEfrags

PROGRAMS

 

• UnSpecific Positive Probe (USPP) filter

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