Matt:LabNotes/2015-4-13: Difference between revisions

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(Created page with "==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_...")
 
>Mzcai
 
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===SeqRolony Gene Counts vs DARTFISH===
===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


===SeqRolony Probe Counts vs in vitro V4 Capture===
====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]]

Latest revision as of 07:35, 15 April 2015

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]

CountReadsPer_Gene_Probe.pl

  • 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

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

File:GNG4 ProbeCount Regression.png