This is a guide to identifying, extracting and plotting schistosome lifecycle microarray data from Fitzpatrick et al., 2009
1. Find your transcript ORF nucleotide sequence from your previous research
2. Open a tab to the IBERS BLAST server this is only accessible on university internet connections
3. On the webpage select the following: For "Program" select BLASTn search, for "Database" select microarray.fasta
4. Paste your transcript ORF nucleotide sequence into the box under the line "Enter sequence below in FASTA format" - this is your query
5. Click the "search" button
6. View and interpret your results page to determine if your transcript if present on the microarray and what it's contig name is
Here is an example BLAST results using SmVAL4 as the query showing a very high e-value - 0.0 for one contig (CONTIG6658) with no other contig having an e-value less than e-20.
Further down the result page, you will see the pairwise alignment between your query sequence (SmVAL4 in this example) and the subject sequence (as known as the "hit") (CONTIG6658 in this example)
It lists range of information including:
- % identity (100%)
- the number of bases which are part of the alignment (506bp)
- whether the strand for the query and subject are the same (not the same in this example)
- which nucleotides differ
7. Click on the subject contig name of your best match which is coloured blue (in this example "CONTIG6658")
8. Copy the resulting sequence into a word document for usage later
Lifecycle Microarray results plotting
9. Download "Dataset S4" from the Fitzpatrick et al., 2009 paper - this is listed under "Supporting Information"
10. Open the Dataset S4 in excel and search using "find" for the Contig name that represents your transcript on the microarray (for SmVAL4, its CONTIG6658)
11. Select this row of data and copy it to a new excel workbook. Also copy the headings for each of the columns into your new workbook.
12. Each lifestage (e.g. egg, miracidia...) has 3 biological replicates (with the exception of the 7wk female, which has 2 replicates), so averages should be plotted for each lifestage and standard deviations for each lifestage used for error bars. Use excel to produce a bar graph representing the lifecycle expression profile of your transcript.