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

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
Line 37: Line 37:


===SeqRolony Probe Counts vs in vitro V4 Capture===
===SeqRolony Probe Counts vs in vitro V4 Capture===
*Within a certain gene, how does distribution of captured padlock probes correlate between DARTFISH (in situ Rolony Sequencing) and in vitro capture
*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]]

Revision as of 07:20, 15 April 2015

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

 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

CountReadsPer_Gene_Probe.pl

  • SeqRolony_BA8V4_Genecounts.txt
  • SeqRolony_BA8V4_Probecounts.txt

SeqRolony Gene Counts vs DARTFISH

  • 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

  • Within a certain gene, how does distribution of captured padlock probes correlate between DARTFISH (in situ Rolony Sequencing) and in vitro capture
  • 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

  • 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