Matt:LabNotes/2015-4-13
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Sequenced Rolony Analysis[edit]
- 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[edit]
- 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
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[edit]
- SeqRolony_BA8V4_Genecounts.txt
- SeqRolony_BA8V4_Probecounts.txt
SeqRolony Gene Counts vs DARTFISH[edit]
- 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
SeqRolony Probe Counts vs in vitro V4 Capture[edit]
- 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
- Correlation should not be very high because in vitro capture is of gDNA
File:Invitro vs SeqRolony ProbeCount Regression.png
Probe Count Correlation per Gene[edit]
- 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
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
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
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[edit]
- 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
- High detected gene: GNG4