Alpheus Demo

Take a tour through a working instance of the Alpheus website populated with Solexa data generated at NCGR by clicking the links below or to the right. The following steps will guide you through some of the most relevant tools available in Alpheus that allow the researcher investigate gene variants and their expression levels in a couple of demo samples generated at NCGR.

Search Sequence Variants
Search to find genes with variants of interest.
  • Click on "Search Sequence Variants" (above or on the right panel).
  • You will see all the samples pertinent to your project listed under List 1 and List 2. For now simply choose Sample1 under List 1 (highlight it by clicking on it). Don't choose any sample from List 2 (we will focus on Sample1 for the time being).
  • Leave the default values in all other panels, and click on "Find Genes". This will return a list of genes in which non-synonymous SNPs were found in at least 4 reads of Sample 1 and at least 30% of the total reads covering the SNP position in all selected samples call the variant.
  • The Query Results Page appears. From the left most "Gene" column, click a gene name to view data for that gene.
  • The Gene Detail Page for that gene appears. Under "Sequence Details", visualize reads aligned to the transcript by clicking a sample name in the "View Pileups" column (all the way to the right). This will launch a the pileup visualization that shows reads and sequence variants detected in this transcript in the selected sample. Small arrows indicate reads in the sample that have been aligned to the transcript.
About the pileup visualization
  • Variants are filtered according to the original parameters used in the gene search. To see all variants without any filtering, click the blue "CLEAR" text.
  • Reapply filters to dynamically see the effects of filtering. Enter "4" reads in the top most text box (Filter where at least [4] reads call variant). Press "Enter" or "Return" on your keyboard, or click "Update". Toggle other filters to see the effects of each filter.
  • The closeup view shows a 1000bp section of the transcript. View a different section of the transcript by sliding the blue window (in the overview section) to either the left or the right.
Pairwise alignments of reads supporting a variant call
  • View all pairwise alignments supporting a variant call by clicking directly on a red, blue or black hash mark that appears on one of the arrows in the closeup pane. A new browser window will appear (Firefox users must allow popups for this site) that displays all pairwise alignments. Read sequences are decorated with colors to show the sequence quality scores of each base.
  • Statistics of the alignment are also shown in this window, like number of perfect matches and percentage of identity.
  • Close the alignment window and the Flash visualization window. Return to the Gene Detail Page.
  • Under "Sequence Details", click on the transcript name in the left most column (e.g. NM_#). The Transcript Detail page appears.
  • Scroll down to see the "Single Nucleotide Variant Analysis Results" table. Only variants that meet the original filtering criteria from the gene search will be displayed. To see all variants without any filtering, click the "Clear" button in the "Restrict Variant Counts" box above, and scroll down to see all the variants that are found.
  • Reapply filters to see the effects of filtering. Click the "Update" button to see an updated table after changing filter parameters.
  • At the bottom of the page there are conventions on how to interpret data in this table.

Search Gene Expression
Search genes by expression as measured by read abundance. This tool is similar to the "Search Sequence Variants" tool, with the difference that here the researcher is analyzing differential expression of genes accros different libraries (or samples). Before, it was possible to focus on one single sample (as we did in the example presented above), but now it is necessary to input at least two samples in order to compare them in terms of gene expression.
  • Click on "Search Gene Expression" (above or on the right panel).
  • Again, you will see all the samples pertinent to your project listed under List 1 and List 2. Since the goal here is to analyze differential expression of genes accross different samples, it would not make sense to choose the same sample in both lists. Choose Sample 1 under List 1 and Sample 2 under List 2, and input 500% in the box above the two lists. With these settings Alpheus will look for genes whose expression level (measured by the number of reads that hit the gene) in Sample 1 is at least five times higher than their expression in Sample 2. Leave the default in all other values, and click "Find Genes".
  • A Query Results Page appears which is very similar to the one described in the "Search Sequence Variants" section. The only difference is that now each list of samples has its own read abundance column, which allows the user to compare gene expression levels between samples accross the two lists. When two or more samples are chosen in one of the lists, the two right most columns show average abundance of reads hitting the corresponding gene in all the samples of the list.
  • All other columns in the table are similar to the columns that the "Search Sequence Variants" tool yields.

Case Overview
View sample read and alignment statistics.

Find Genomic Span
View sequence variants within a 10Kbp span by entering genomic coordinates.

Project Data

Search Sequence Variants

Search Gene Expression

View Case Overview

Find Genomic Span

Release Notes