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_...")
 
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===SeqRolony Gene Counts vs DARTFISH===
===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===
===SeqRolony Probe Counts vs in vitro V4 Capture===

Revision as of 06:05, 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