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==RNA in situ sequencing== ===Responsibilities=== *Rui: in situ rolony generation *Matt: design and testing of decoding padlock probes *Ho Suk: instrumentation (fluidic devices), automation, image analysis ===Method developments=== ====Probe design==== *Strategy: Experimentally evaluate the capture efficiencies of 6,500 probes(or 13,000, depending on the minimal order allowed by Agilent), and pick the best ones. *Choice of genes: #Variable expression among different brain regions and cell types (based on published [http://www.nature.com/nature/journal/v489/n7416/full/nature11405.html ABI Brain Atlas data]). #Moderate abundance (too low: difficult to detect; too high: rolonies too crowded and difficult to resolve optically). #Avoid alternatively spliced exons. #Prioritizing genes used in [http://www.sciencedirect.com/science/article/pii/S0092867412003480 ABI ISH mapping]: Disease genes (383); Human cortical marker genes (167, 55 overlapped with disease genes). *Considerations: #Targeting RNA versus cDNA (RNA: one fewer step, more specific, efficiency could depends on secondary structure; DNA: one more step, can be immobilized, secondary structure could be less a problem); #Targeting nuclear transcripts versus cytoplasmic transcripts (space available for imaging, integration with single-cell toto-RNAseq data), nucleus versus cytoplasmic transcripts (based on the [http://www.nature.com/nature/journal/v489/n7414/full/nature11233.html ENCODE RNAseq paper]). #Targeting 3'-ends or random positions (affects how cDNAs are generated, poly-T primed or N6/9 primed); #Gap versus no gap (Specificity versus sensitivity); #Targeting single exons or across splice junctions (mature transcripts versus nascent transcripts or gDNA); *Constrains: #We can get only up to 240bp oligos from Agilent for now. #A minimal list of features on the probes: ##Amplification adaptors (36-44bp, depending on the probe prep protocol). ##Common linker (48bp for our standard linker sequence, need to be reduced significantly, which is fine since we will be doing RCA instead of PCR and only a short annealing region is enough) ##Decoding region (6x20bp=120bp) ##Capturing arms (40-50bp, need to set an upper limit) *First round probe design. **A list of [[Media:Gene-selection-top5k.xlsx|5333 genes]] were generated by Dr. Yun Zhu from Wei Wang's group based on the following criteria: ***Genes that show variable expression in the brain (based on PMID: 22996553). ***Genes included in the [http://www.cell.com/retrieve/pii/S0092867412003480 ABI's ISH data]. ***Genes present in both cytosol and nucleus (based on the [http://www.nature.com/nature/journal/v489/n7414/full/nature11233.htm ENCODE RNAseq paper]). **Matt selected up to 5 candidate regions per gene for probe design. One set of probes was designed with a gap size of 20bp. A second set has no gap. ***[[Media:outputFile_20gap.txt|Raw probe sequences (20bp gaps)]]. ***[[Media:outputFile_0gap.txt|Raw probe sequences (0bp gaps)]]. **I wrote [[Media:filter_probes.txt|a script]] to filter the probes to keep: ***Up to 5 probes per gene. ***All genes with names like "LOCxxxx" or "CxxOrfxxxx" are excluded because they are poorly annotated. **I then double-checked these probes by mapping to the human genome and reference mRNA sequences using NovoAlign. All probes that were not mapped to the refMrna, or were mapped to more than one location in the genome are excluded. [[Media:probes2fasta.txt|./probes2fasta.pl]] < outputFile_0gap_filtered.txt > outputFile_0gap_filtered.fasta [[Media:probes2fasta.txt|./probes2fasta.pl]] < outputFile_20gap_filtered.txt > outputFile_20gap_filtered.fasta /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/RNAseq/Data/CommonFiles/refMrna.ndx -f outputFile_0gap_filtered.fasta -F FA -r ALL > outputFile_0gap_filtered_novoalign_refMrna.out /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/softwares/Novocraft/novocraft/human_g1k_v37 -f outputFile_0gap_filtered.fasta -F FA -r ALL > outputFile_0gap_filtered_novoalign_human_g1k_v37.out & /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/RNAseq/Data/CommonFiles/refMrna.ndx -f outputFile_20gap_filtered.fasta -F FA -r ALL > outputFile_20gap_filtered_novoalign_refMrna.out /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/softwares/Novocraft/novocraft/human_g1k_v37 -f outputFile_20gap_filtered.fasta -F FA -r ALL > outputFile_20gap_filtered_novoalign_human_g1k_v37.out & *For 83 stem cell genes that no good probe was designed in the first pass, Matt repeat the probe design using mRNA sequences as the template. He generated two additional probe files, one for 20bp gaps, and one for 0bp gaps. I did the mapping to refMrna and human_g1k_v37. ====Linker design==== *I wrote a [[Media:get_decoding_linkers.txt|script]] to generate a list of barcoded linker sequences. A total of [[Media:decoding_linkers_8-stages_2-colors.txt|1792 linkers]] were generated using two colors and eight stages. *Since we will start with ~5000 candidate genes, more barcoded is needed. [[Media:get_decoding_linkers_3_colors.txt|For this I increased the number of colors (fluorophores) to 3]]. With only 7 hybridization stages, we can decode [[Media:decoding_linkers_7-stages_3-colors.txt|5040 genes]]. I also added another constrain that each barcode needs a minimal of two colors. The barcodes are also shorter, which gives more flexibility for the design of the common linker. ====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]] ====Rolonies generation==== *Considerations: #Direct padlock capture versus whole transcriptome amplification
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