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==Sequenced Rolony Analysis== *Library prep: [[Matt:LabNotes/2015-3-31]] **Sequences are from CA12kNov2014_V4 Rolonies in BA8 tissue *Raw data: /home/kunzhang/seqStore/150411_MiSeq/MC-SeqRolony_BA8V4_Mar3115-13_CCATGA_L001_R1_001.fastq *Working directory: **Genome-miner: BA8_V4_RolonySequencing -> /media/LTS_15T/MC_LTS/InSitu_MiSeq_150411_Analysis/ **Local: C:\Users\Matt\Dropbox\GradZhangLab\CA12k_Nov2014\SeqRolony ===Mapping to Probelist=== *Base quality /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i MC-SeqRolony_BA8V4_Mar3115-13_CCATGA_L001_R1_001.fastq -o MC-SeqRolony_BA8V4_Mar3115-13_CCATGA_L001_R1_qualstats.txt /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC-SeqRolony_BA8V4_Mar3115-13_CCATGA_L001_R1_qualstats.txt -o MC-SeqRolony_BA8V4_Mar3115-13_CCATGA_L001_R1_qualstats.png -t SequenceRolony_BA8_V4 [[File:MC-SeqRolony_BA8V4_Mar3115-13_CCATGA_L001_R1_qualstats.png|550px]] bowtie2-build CA12k_Nov2014_V4_H1H2.fa CA12k_Nov2014_V4_H1H2 bowtie2 --phred33 -x CA12k_Nov2014_V4_H1H2 -q MC-SeqRolony_BA8V4_Mar3115-13_CCATGA_L001_R1_001.fastq > SeqRolony_BA8V4.sam 2> SeqRolony_BA8V4_stderr.txt & 351727 reads; of these: 351727 (100.00%) were unpaired; of these: 16931 (4.81%) aligned 0 times 334796 (95.19%) aligned exactly 1 time 0 (0.00%) aligned >1 times 95.19% overall alignment rate samtools view -bS SeqRolony_BA8V4.sam | samtools sort - SeqRolony_BA8V4_sorted samtools view -h -F 4 SeqRolony_BA8V4_sorted.bam > SeqRolony_BA8V4_sorted_filtered.sam ===Count Probes and Genes=== [[Media:CountReadsPer_Gene_Probe.txt | CountReadsPer_Gene_Probe.pl]] *SeqRolony_BA8V4_Genecounts.txt *SeqRolony_BA8V4_Probecounts.txt ===SeqRolony Gene Counts vs DARTFISH=== *[[Media:RCommands_SeqRolonyAnalysis.txt | R Commands]] *Good correlation suggests Rolony Sequencing experiment was successful (despite leaking/evaporation during slide PCR and needing to "resuspend" with H2O) **Rolony Sequencing counts can be used to estimate DARTFISH counts [[File:DARTFISH_vs_SeqRolony_Regression.png|550px]] ===SeqRolony Probe Counts vs in vitro V4 Capture=== *Compare probe counts between Rolony Sequencing and in vitro **Correlation should not be very high because in vitro capture is of gDNA ***If correlation is high, that means differences in gene expression can not be detected [[File:Invitro_vs_SeqRolony_ProbeCount_Regression.png|550px]] ====Probe Count Correlation per Gene==== *Within a certain gene, how does distribution of captured padlock probes correlate between DARTFISH (in situ Rolony Sequencing) and in vitro capture **Do one gene at a time to negate influence of different transcript (target) abundance in tissue *High detected gene: PDE1A [[File:PDE1A_ProbeCount_Regression.png|550px]] PDE1A_TTTGAacacacacacacacacacacacacacacacacacaTTCTTGTCTC PDE1A_CCTCAGCATCCCACCTGTCATTCTGTTCCCATGCATTTAAAATAAG PDE1A_CCTCATCTTCTGCACATGTATTGCACAGTCATTATGACAGGGTTCAAAGC PDE1A_AAGAGCCATAGCTTTGTGGAAAGTTGAGAATTTAGGATGTTTTCATGCTGA PDE1A_TCTTTAAGGTGTTTCGGGCCTATGAATGTGTCTCATCATGTTTTTCTTCAG PDE1A_CTGAAAGCACAAGGTGCTGATGTAGCCACTAGATGAATCTGTTCGGTAGC PDE1A_TCTTTCCACCTCTCTTTGTTCTGCTGAATGATGTCCACCAGGTTGTTCTTG PDE1A_TCGGCTTTTGAGGCTTCCTCTATAAGAGGAATAACAATTTTCTCTGTTGAG PDE1A_ATGGTTGACTTCCGATCACAAAGTGGGGAAAATGGAAGCCCTAATTCAGCT PDE1A_GAAAAACTCCTCCATTAGGGCCATGGTCCACCGATAATGCAGCTTCC PDE1A_AGGCTGCTGCAAACTGTTTCTTATATTTTTAATTTGCTGGAAGTGACCTGA PDE1A_ATCAAGATATTCATTTCTTCTTCTTGCATAAGTCGATAAGCTGCACTCAC PDE1A_TGTGAAAGTTGTTTGTTGTCCCTGTATGCTCATAATCATGAATGGCAGCAG PDE1A_TTTGAGTGACATCAGCTGCATGAATCAAATTGTGATATGGATTTTTGTACT PDE1A_AGACTATGCTCTCCACTTGCTTCATTTAGGGCAAATACATCGAAAGACCAT PDE1A_ACTGAGTCAGTCTGAATGTCACTGAGCTCATCTTCAGTATCCAGAAGTCTT PDE1A_AGCACAGATGCCGCATATTCAATATTCTTCTTTAAGTCGACGACGTTAACA PDE1A_TCTTCAATCTCTGTGGCACTAGACCCCATGATGATGCTCCTAAGACAATAC *Medium detected gene: MALAT1 [[File:MALAT1_ProbeCount_Regression.png|550px]] MALAT1_CCTTCTGAACCGGAGCAGGAAGAAAAATAAAAGCTTTCCTCCAAAC MALAT1_CGCCATGGAAAGCGAGTTCAAGTGGCCTTTTAAAGTAGACCAACTA MALAT1_CCTCACAAAACCCCCGGAACTTTTAAAATACCTCTTAAAGCACTTC MALAT1_ACTCCATGAAGAAGCTTCATCTCAACCTCCGTCATGTTTTAGAAACCTTT MALAT1_GGCTTTAAATGACGCAATTCTCCCTGCGTCATGGATTTCAAGGTCTTTTAA MALAT1_TTCTGGTCTACGTAAACACCCTCATCTTCTCAAGCTTTACCTTCTAACTTC MALAT1_GCTACCTTCATCACCAAATTGCACTCGCTCCTTCCTGGAATCCTTTTTCC MALAT1_TCATGTAGTCCACCTGAACATCAACAGTTCAGCCTCTACAGAGTTTCTGT MALAT1_GCATCTGCTGGTTCGCTTGATCTGAGTTAAAATGAAACAAAGGCTT MALAT1_TGAGTCAAGCCCGGTGAGTATTCACTTTTGATCACTATTTTCTCCA MALAT1_CTCTCCTGGCTCCTTCTTTACCTCCTTTAAGCCCATGTTAAACAGGCCAT MALAT1_AAACACACGATCTGGGGGTTAGATCAAGAATTCAGGGATTGTCCAGCAAG MALAT1_GTCTGGACTCGCAATAAGCAATCTGGTTCTATCTCCTGTATTTGCTCCGC *Low detected gene: NEFL [[File:NEFL_ProbeCount_Regression.png|550px]] NEFL_TGCACATAGCTTGCATCTGTTTGAGACTTACCATGTACATCAACCCAGGT NEFL_GGATACATGGGGCGTGTATTTGATGCAATGTCCAACCAGTCAAGCTATCA NEFL_CTCCCTAGAAAGCCATCCCAAAACCTAACACAACACGTGTTATGAGGCAA NEFL_CCACTCTGCAAGCAAACAGATACTCTGCATAAAGAGGAAATTCATAGCAC NEFL_ttcagcttctttggtttcctctccttcttcaccttcacctccttcttcttc NEFL_GTTTCCTCCACTTCGATCTGCTCCTCTTGGACATGGCTGGTGTAGTAG NEFL_ACGTTGAGGAGGTCTTGGTATTCTTTTAGGTATCGTGCCATTTCACTCTTT NEFL_GGTGAAGCGGCTCTTGAACCATTCCTCAGCGTTCTGCATGTTC NEFL_CTACCTGGCTCAGGTCGAGGTTCTCCAGACTGGGCATCAAC ====Try some other high detected genes in DARTFISH==== *See if correlation is only good for highly detected genes in DARTFISH **Low DARTFISH counts are more affected by stochastic variation *High detected gene: KCNC2 [[File:KCNC2_ProbeCount_Regression.png|550px]] *High detected gene: GNG4 [[File:GNG4_ProbeCount_Regression.png|550px]]
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