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]

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