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====Probe assembly==== *The 0bp set used AP1V6/AP2V6 primers for amplification. *The 20bp set used AP1V4/AP2V4 primers for amplification. [[Media:probe2padlockFISSEQ_2012_0bp.txt|./probe2padlockFISSEQ_2012_0bp.pl]] > [[Media:Dec2012_0bp_gap_probes.txt|Dec2012_0bp_gap_probes.txt]] [[Media:probe2padlockFISSEQ_2012_20bp.txt|./probe2padlockFISSEQ_2012_20bp.pl]] > [[Media:Dec2012_20bp_gap_probes.txt|Dec2012_20bp_gap_probes.txt]] $ wc -l Dec* 12344 Dec2012_0bp_gap_probes.txt 12511 Dec2012_20bp_gap_probes.txt 24855 total *I made some modification on the assembling scripts for the probes that were designed based on mRNA sequences. The reason being that they are multiple transcripts per genes, and some probes designed for different isoforms could be identical or overlapping. [[Media:probe2padlockFISSEQ_2012_0bp_stemCellGenes.txt|./probe2padlockFISSEQ_2012_0bp_stemCellGenes.pl]] > [[Media:Dec2012_0bp_gap_probes_stemCellGenes.txt|Dec2012_0bp_gap_probes_stemCellGenes.txt]] [[Media:probe2padlockFISSEQ_2012_20bp_stemCellGenes.txt|./probe2padlockFISSEQ_2012_20bp_stemCellGenes.pl]] > [[Media:Dec2012_20bp_gap_probes_stemCellGenes.txt|Dec2012_20bp_gap_probes_stemCellGenes.txt]] wc -l Dec2012_stemCellGene_* 785 Dec2012_stemCellGene_0bp_gap_probes.txt 843 Dec2012_stemCellGene_20bp_gap_probes.txt 1628 total *I merged all assembled probe files. It turned out that there are still some redundant sequences in the first batch of brain gene probes. So I used the Excel "Remove Duplicates" function to remove all redundant sequences. This yielded a final list of [[Media: FISSEQ_Probes_Dec2012.txt|26,143 probes]] (228bp in length) that was submitted to Agilent for synthesis. =====Feb. 2013 set===== *Agilent has kept delaying the oligo synthesis. The current estimated delivery date is March 14th. There won't be enough time to meet our NIH deadline on 3/31. *I just learned that another company, CustomArray Inc, can make up to 200mers, and the turnaround time is a week. So I decided to order some oilgos from CustomArray to get things going. The constrain here is the length, which is only 200nt. This is already on the long end for CustomArray, and they are not very certain about the quality. *The plan is get one 12k library, and give it a try. Will test the no-gap probes first, because they are likely to be more efficient. *To reduce the probe size to 200nt, I have to find ways to trim different parts of the probes. #Reduce the length of each decoding oligo from 22nt to 20nt. Also need to eliminate the oligos (5_3, 7_1) that can be cut by the nicking enzymes. [[Media:get_decoding_linkers_4_colors.txt|get_decoding_linkers_4_colors.pl]] #Replace the common amplification adaptors to the short version in Porreca et al NMeth 2007. That means the libraries should be amplified with eMIP_CA1/eMIP_CA2 primers, and the functional probes will be released using nicking enzymes. #Reduce the total H1/H2 length to 46. #Reduce the length of the common linker by trimming the 3'-ends to: CTTCAGCTTCCCGATATCCGACGG. [[Media:probe2padlockFISSEQ_Feb2013_0bp.txt|probe2padlockFISSEQ_Feb2013_0bp.pl]], [[Media:probe2padlockFISSEQ_Feb2013_0bp_stemCellGenes.txt|probe2padlockFISSEQ_Feb2013_0bp_stemCellGenes.pl]] *With all these changes, I ended up getting 11754 probes for 3840 brain genes ([[Media:Feb2013_0bp_gap_probes.txt|Feb2013_0bp_gap_probes.txt]]), and 759 probes for 84 stem cell genes ([[Media:Feb2013_0bp_gap_probesstemCellGenes.txt|Feb2013_0bp_gap_probesstemCellGenes.txt]]). After merging the two sets, 178 duplicated oligos were found and removed, leaving 12,335 probes to be submitted for synthesis:[[Media:Feb2013_200bp_probes_to_order.txt|Feb2013_200bp_probes_to_order.txt]]
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