High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity
© Zhou et al; licensee BioMed Central Ltd. 2009
Received: 22 January 2009
Accepted: 5 October 2009
Published: 5 October 2009
Commercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity.
The human Genome-Wide SpliceArray™ (GWSA), a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC) Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform.
Significant changes were detected independent of transcriptional activity, indicating that the controls for transcript generation and transcription are distinct, and require novel tools in order to detect changes in specific transcript quantity. Our results demonstrate that the SpliceArray™ design will provide researchers with a robust platform to detect and quantify specific changes not only in overall gene expression, but also at the individual transcript level.
A large portion of the diversity within the transcriptome is generated by alternative splicing, which in some cases, can produce thousands of transcripts from a single gene or locus [1, 2]. This has important implications in biology and pathophysiology where extensive alterations in transcripts resulting from alternative splicing produce structurally different products and impacts the function of genes in biology, disease [3–6], as well as processes such as evolution [7, 8]. The fine granularity of the transcriptome has not been determined with clarity, and new commercial tools are required in order to begin to identify with certainty the diverse content of the transcriptome. Traditional microarray designs and analytical methods are not robust enough to detect this transcript diversity. SpliceArray™ microarrays were developed to experimentally define the composite of transcripts that are present within biological samples and have the ability to detect subtle differential changes in gene expression for different alternatively spliced isoforms. The performance parameters of the human Genome-Wide SpliceArray™ (GWSA) which monitors over 280,000 potential splicing events (known and predicted) were assessed here, guided by the recent publications from the MicroArray Quality Control (MAQC) consortium.
The MAQC project  is a FDA sponsored consortium founded to address concerns of microarray reproducibility of expression profiling experiments. The purpose was to generate quality control tools for the microarray community in order to avoid procedural failures and to develop guidelines for microarray data analysis by providing the public with large reference datasets along with readily accessible reference RNA samples. The study found, that overall, the platforms perform similarly  and were validated with alternative quantitative gene expression platforms . However, all the platforms tested contain a similar bias where probes were designed to monitor the overall level of the gene, and do not give any expression information toward the isoform diversity produced from each gene through alternative splicing. Here we present a novel microarray platform and analysis for the efficient detection of isoform diversity on a genome wide scale in human. Our analysis of this new microarray design is in accordance with the approach outlined by the MAQC consortium and demonstrates that the SpliceArray™ products are highly reproducible, quantify transcripts, and are sensitive in detecting subtle changes in transcript ratios.
Distribution of splice events from an analysis of the human genome.
Number of events
Total putative alternative splicing events with cDNA supporting evidence:
▪ novel exon
▪ novel exons
▪ exon skipped
▪ exons skipped
▪ alternative splice donor (ASD)
▪ alternative splice acceptor (ASA)
▪ intron retention
▪ novel intron
Predictions of single exon skips with no supporting cDNA evidence
Intron/exon boundary regions with structural probes for prediction of ASA, ASD and intron retentions
To assess performance of the human GWSA, samples were selected in accordance with the MAQC project, which included human universal reference RNA (Sample A), human brain RNA (Sample B) and two titration samples (C and D; see methods; refer to fig. 1 in ). As with the MAQC project , we assumed a linear response for each probe across the four titration samples (A-D). The frequency distribution of the expression values for all 12 arrays (samples A-D run in triplicate) showed a normal distribution of signal as expected for the platform (Additional file 2-A). The reproducibility of the arrays was found to be highly concordant and produced low coefficients of variation (CV%) for each sample analyzed. The mean CV% were found to be slightly higher than the median values (5.5 versus 4.23, respectively), indicating a slight bias towards higher values. On a gross level, principal component analysis (Additional file 2-B) showed close grouping of the replicates.
Correlation of gene level analysis between the human GWSA and the Affymetrix HG-U133 Plus2.0 array.
# of Genes represented
1.5 Fold Change (FC)
Event analysis of genes grouped by overall gene expression.
Gene Expression Level
(median of F and T)
Fold Change Set
Subset of events with ≥3 Fold Change
x ≥ 3 or ≤ -3
-1.2 ≤ x ≤ 1.2
Analysis of gene expression has provided researchers with an important resource to identify genes involved in different biological processes, and has been used to generate profiles where the expression level of a predefined set of genes can help to identify and predict a variety of pathological states and prognoses [14, 15]. However, many of these studies have ignored the large diversity of transcripts that are generated from each locus by alternative splicing and miss the rich source of diverse transcripts. Researchers have treated each gene as a single entity which is inaccurate considering that potentially over 90% of genes undergo alternative splicing as evidenced by our analysis and recent reports [16, 17]. In an attempt to identify different transcripts, studies have been performed where probes have been reordered into new probe sets to define transcripts more accurately [18–20]. However these studies suffer from the same flaw, that the original design of the microarray was not focused on detecting alternative transcripts per se, but to provide an accurate measurement of the overall expression of each gene, not transcript. In contrast, the human Genome-Wide SpliceArray™ (GWSA) is designed to detect alternatively spliced events through the use of exon body and junction probes.
Since Johnson and co-workers  published the first human genome-wide microarray that utilized exon-exon junction probes, several groups have simultaneously produced microarrays to monitor splice events using similar probe designs [11, 22–24]. The GWSA microarray is the most comprehensive design commercially available with over 6 million probes. The array design consists of a maximum of 6 probe types targeted to the exon body, exon-exon junctions, and exon-intron/intron-exon junctions allowing more complete detection of transcripts, in contrast to those commercially available arrays that incorporate only exon body probes  or only junction probes . The SpliceArray™ probe design simultaneously monitors both isoforms for each splice event and is able to detect alternative exon, intron retention, alternative splice acceptor (ASA), and alternative splice donor (ASD) events. Additionally, the design has the ability to monitor putative novel skipped exons and unidentified ASA and ASD events. Although the design is theoretically capable of detecting all types of events, it does have the limitation where very short sequence differences (less than 8 bases) are not captured and these events have been filtered out of the collection. In addition, the microarray platform itself has limited space and even at the maximum number of features, only 6.54 million probes can be included which limits the number of probes per probe set that can be designed for any event whether evidenced or predicted. Furthermore, microarray platforms possess limitations on the sensitivity to detect very small changes in transcript expression. A consequence of manufacturing the GWSA content on the Affymetrix platform is that the cost for updating the array probe content is significant as it would require re-manufacturing a custom array and therefore recent discoveries of novel splice events cannot feasibly be added to the design. However, it should be pointed out that the GWSA monitors ~140,000 predicted exon skip events so although there was no evidence of these exon skip events at the time the GWSA content was generated, it indicates that the array has the potential to monitor newly discovered exon skip events.
Concurrent with the generation of microarray designs able to detect alternatively spliced transcripts, many analytical methods have been developed to determine the extent of alternative splicing and identify the expression level for each transcript within a gene. We used a simple approach to identify cases where the ratio of the inclusive and exclusive isoforms changed dramatically, by calculating the ratio of probes that were specific for the inclusive or long form (probe B) versus probes specific for the exclusive or short form (probe E). This approach was shown to provide very powerful results when assessed against PCR validation of the events. It is a simple and applicable approach that provides strong filtering methods for real positives, unlike other methodologies which require an extensive programming and array fitting .
Performance of the human GWSA product was evaluated in this study and the arrays were shown to be highly reproducible, suggesting that the reproducibility is more a function of the platform and labeling procedures than of the probe design. Analysis of the array data at the gene level proved highly concordant with data produced in the MAQC project on the HG-U133 Plus 2.0 array. Even though the platforms were slightly different (the HG-U133 Plus 2.0 array contains 11 micron features while the GWSA contains 5 micron features), the genes showed similar levels of expression, and importantly, comparable quantified changes between the universal and brain RNA samples. High concordance was observed between these similar platforms even though the probe sets were designed for different applications. Additionally, the GWSA and other SpliceArray™ products also provide splice event information available from the same experiment, exemplified by several recent reports [3, 4, 26–29].
Validation of specific splice events from the GWSA hybridizations was performed based on the analysis of the ratio of the long isoform to the short isoform. This type of assessment is an important aspect to determining the extent of alternative splicing as in many cases the ratio of isoforms will determine the biological response [30–33]. By assessing the statistical power of the ratio, we were able to validate a high rate of events; overall, 81% of statistically selected events were validated by RT-PCR. The high rate of validation was found regardless of the overall transcriptional changes identified. Interestingly, almost 60% of the events identified to have gene level, transcriptional alterations of 3 fold or more were found to have an event fold change of 3 fold or more. This indicates that not only is there a change in overall gene expression, but the transcripts produced are different as generated by alternative splicing. Surprisingly, genes that showed no change in their expression levels represented a rich source of alternatively spliced transcripts. Almost 10% of the events among these genes contained evidence of splicing alterations by changes in the ratio of the different isoforms. This is an important finding as it brings to point that genes which have been ignored because of equivocal expression between samples actually have important changes that occur in producing different isoforms. This confirms earlier evidence  that illustrates transcriptional activity is independent of splicing, even though the two processes function simultaneously and emphasizes the need to measure both overall gene expression and alternatively spliced transcripts for greater understanding of biological processes. The above findings should encourage researchers to look more closely at the genes which show no variation in overall gene expression for important clues into the mechanisms in pathophysiology and biology.
Alternative splicing affects not only the structure of the mRNA but ultimately the structure of the protein produced. Many exons encode protein domains that can be removed by an exon skip event . Such an event can produce proteins that lack a functional domain, dominant negative proteins, constitutively active isoforms, soluble homologues, or can lead to the regulation of the protein's overall activity by altering its global expression. The two examples, erythrocyte membrane protein band 4.1-like 3 (EPB41L3), and Lim domain 7 (LMO7) depicted here show how changes at the transcript level can be observed with or without gene expression changes, respectively. Multiple alternative splice variants have been previously described for LMO7  and the variant detected here results in deletion of the Lim domain, a protein-protein interaction domain found in many key regulators of development. EPB41L3 is potentially a critical growth regulator in meningioma pathogenesis  and is normally expressed at high levels in brain, with lower levels in kidney, intestine, and testis. The function of the EPB41L3 variant is unknown, yet contains some well described protein domains, including Ferm domains found in many cytoskeletal proteins, the N domain found in ubiquitin-like structural domain, and the C domain found in tyrosine phosphatases . Different isoforms certainly will affect the function of these different domains and potentially the protein, and may play a role in pathological states.
The identification of expressed transcripts is at the heart of expression analysis and these examples, as well as recently identified novel spliced isoforms demonstrating diverse activity [4, 6] illustrate the importance of correct determination of the expression of each transcript. Array platforms specifically designed to detect the differences in transcripts along with other novel technologies  will allow researchers to more fully explore the transcriptome under different physiological and pathological conditions. In short, we have provided a unique platform to detect isoform diversity in the human transcriptome by utilizing algorithmically designed novel exon body and splice junction probes to detect every possible isoform event. This approach on a genome-wide scale has been demonstrated to be highly reproducible and quantitative.
Total RNA was purchased from Stratagene (Universal Human Reference RNA, # 740000; sample A) (LaJolla, CA, USA) and Ambion (Human Brain Total RNA, # AM7962; sample B) (Austin, TX, USA). Total RNA quality and concentration were verified using the Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA). Four titration samples (A, B, C = 75%A+25%B, and D = 25%A+75%B) were generated as previously described  and each sample was analyzed in triplicate.
The content for the human Genome-Wide SpliceArray™ (GWSA) was designed using Build 35 of the NCBI Human (genome) reference sequence produced by the International Human Genome Sequencing Consortium and sequence data from the NCBI GenBank sequence database. Additional information was used from the NCBI Entrez Gene database in the representation of the genes. For each gene, a RefSeq was selected as the reference form, the selection considered which RefSeq (when more than one was available) covered the largest genomic area to maximize sequence inclusion (based on mapping against a genomic range overlapping the selected representative). Sequences used in the analysis were aligned to the human genome and data from this alignment was used to create distinct exon structures. Significant differences from the reference sequence were detected and probes were designed to monitor each difference or splice event as previously described . All splice events included on the GWSA were subject to manual review to remove those events that appeared to be indicated by sequence or alignment artifact. Additionally, splice events were removed with the following characteristics: those that resulted in very small changes (of a few nucleotides) to the transcript; where the required probes would not conform to Affymetrix manufacturing standards; and that would not be detected by the SpliceArray™ probe configuration. The human GWSA custom microarrays were manufactured by Affymetrix (Santa Clara, CA, USA) as a 49 format, 5 micron array containing over 6 million probes which monitor approximately 229,000 exons and 181,000 junctions. GC probe sets for background correction and positive and negative control probes were included on the array. The probes on the array are designed in the sense orientation and for use with the NuGEN WT-Ovation™ RNA Amplification Systems and FL-Ovation™ Biotin Module Products. For additional information on SpliceArray products and probe design, refer to http://www.splicearray.com.
MicroArray processing: Transcript amplification and labeling
Amplified cDNA was prepared using the WT-Ovation™ Pico RNA Amplification System (NuGen product #3300; Santa Clara, CA, USA) starting with 50 ng of each total RNA sample, as described by the manufacturer. Briefly, the RNA is synthesized into double-stranded cDNA using a SPIA™ DNA/RNA primer. The DNA portion of the primer binds poly (A) sequence or randomly across the transcript, and the RNA portion of the primer is incorporated into an unique DNA/RNA heteroduplex at one end of the cDNA. RNase H degrades the RNA portion of the DNA/RNA heteroduplex allowing for additional SPIA™ primer to bind and initiate replication by DNA polymerase. The process of SPIA™ DNA/RNA primer binding, DNA replication, strand displacement and RNA cleavage is repeated resulting in isothermal linear amplification of the cDNA. On average, the cDNA yield from 50 ng of high quality total RNA ranges from 6 to 10 micrograms. Fragmentation and biotin-labeling of the amplified cDNA was performed using the FL-Ovation™ cDNA Biotin Module V2 (NuGen product #4200) as per manufacturer's instructions.
Array hybridization, scanning, and data extraction
Prepared target for each sample was hybridized to the Affymetrix-formatted SpliceArray™ product using standard hybridization methods recommended by the manufacturer for the 49-format (Affymetrix - Expression analysis technical manual). The arrays were stained and washed using the Affymetrix FS450-0001 fluidics protocol prior to scanning with the Affymetrix GeneChip® Scanner 3000 7G. DAT and CEL images were visually inspected for anomalies and accurate grid placement. All data are available through the Gene Expression Omnibus (GEO) database as series accession number GSE17258.
The analysis described here was generated using Partek® software (http://www.partek.com/). For the comparative analysis, data for the HG-U133 Plus 2.0 GeneChip was taken from the MAQC data set publicly available at http://edkb.fda.gov/MAQC/MainStudy/upload/. All array data was pre-processed which included probe level RMA background correction, quantile normalization across all arrays, and Log2 transformation followed by median polish to summarize probes to obtain the overall score for each probe set. The data were filtered based on the expression values of each probe set within the triplicate set for each sample; if the expression value of a probe set was below 3.5 (log 2 value) the probe set was removed from the analysis. In order to identify probe sets that were significantly different between samples A and B, a one-way ANOVA statistical test was performed on normalized and filtered probe set level intensity values between each group to generate p-value and fold change data.
To identify genes that were significantly different between samples A and B, a list of non-redundant evidenced probe sets were imported into Partek® software. Non-logged median values for F and T evidenced probe sets with the same Entrez Gene ID were calculated to generate an overall gene score for each gene. After removing genes which were expressed at a low level, a one-way ANOVA statistical test was then performed on the gene level intensity values between sample groups A and B to generate p-values and fold changes for each gene.
In order to estimate gene level correlation between the human GWSA and the HG-U133 Plus 2.0 array data, both data sets were imported separately and subjected to the same pre-processing method. For the GWSA data, a gene score for each sample was generated as described above. After separately filtering the low-expressing genes in both data sets, a total of 7,147 common genes were identified. The log value of the ratio of sample A/sample B of gene values was calculated for each gene and platform, and a linear correlation coefficient was calculated between the GWSA and HG-U133 Plus 2.0.
A one-way ANOVA statistical test was subsequently performed on the log 2 based ratios between each of the sample groups to generate p-values and fold change of the B/E ratio between samples A and B. Similar methods were used to calculate C/E and D/E ratio, which provided similar data.
Using the human GWSA gene level intensity data, two lists were generated, those genes that displayed no differential expression change (-1.2 ≤ X ≤ 1.2) between samples A and B and a second group showing significant gene expression change, greater than 3 fold change. ANOVA analysis identified events within these two separate groups having significant B/E ratios between samples A and B. Top events for RT-PCR validation were selected by choosing events with the highest fold changes, p-values < 0.001, and that titrated all 4 samples correctly.
First strand cDNAs were prepared using a random priming protocol starting with 5 μg of the individual RNA samples using the High Capacity cDNA Archive Kit (Applied Biosystems; Foster City, CA, USA). cDNA was prepared from both the human normal brain and universal human reference RNA samples and the quality and consistency were assessed by determining the levels of ribosomal protein P0 by RT-PCR. Additionally, samples were normalized by the level of ribosomal P0 for validation of each splice event.
Lasergene/Primer Select (DNAstar) software was used for primer design. Primer sequences will be provided upon request. RT-PCR amplification was performed using a cDNA dilution with the equivalent of 17 ng of total RNA for each reaction. Promega 2× PCR Master Mix (Madison, WI, USA) was used for each 50 ul reaction with forward and reverse primer at a final concentration of 0.3 uM. A "touchdown" cycling program was used to amplify products (annealing temp 65°C-60°C, 5 cycles, and 60°C, 35 cycles). 12 μl of PCR products were analyzed using either 2% Agarose or 3% Metaphor gels.
Partek® Genomic Suites was used for all analysis. Copyright Partek and all other Partek Inc. product or service names are registered trademarks or trademarks of Partek Inc., St. Louis, MO, USA.
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