Editing
Matt:LabNotes/2017-1-3
(section)
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
=CA12k_Oct2016_V4 in vitro Capture Sequencing Analysis== *[[Matt:LabNotes/2015-7-12 | For reference doing analysis]] *[[Matt:LabNotes/2016-11-15 | in vitro capture experiment]] **Indx1: T4 **Indx2: SplintR **Indx3: Ampligase ===Check Sequencing Quality=== *Currently in genomeMiner:~/scratch/CA12kOct2016_V4_CaptureAnalysis/Matt /media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i Index1_S1_L001_R1_001.fastq -o MC20161215_CA12kOct20161215_V4_T4_Indx1_qualstats.txt /media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i Index2_S2_L001_R1_001.fastq -o MC20161215_CA12kOct20161215_V4_SplintR_Indx2_qualstats.txt /media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i Index3_S3_L001_R1_001.fastq -o MC20161215_CA12kOct20161215_V4_Amp_Indx3_qualstats.txt /media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20161215_CA12kOct20161215_V4_T4_Indx1_qualstats.txt -o MC20161215_CA12kOct20161215_V4_T4_Indx1_qualstats.png -t CA12kOct2016_V4_T4 /media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20161215_CA12kOct20161215_V4_SplintR_Indx2_qualstats.txt -o MC20161215_CA12kOct20161215_V4_SplintR_Indx2_qualstats.png -t CA12kOct2016_V4_SplintR /media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20161215_CA12kOct20161215_V4_Amp_Indx3_qualstats.txt -o MC20161215_CA12kOct20161215_V4_Amp_Indx3_qualstats.png -t CA12kOct2016_V4_Ampligase [[File:MC20161215_CA12kOct20161215_V4_T4_Indx1_qualstats.png | 650px]] [[File:MC20161215_CA12kOct20161215_V4_SplintR_Indx2_qualstats.png | 650px]] [[File:MC20161215_CA12kOct20161215_V4_Amp_Indx3_qualstats.png | 650px]] *Acceptable quality scores up to 65bp read, after 65bp quality drops ===Mapping Reads to Probelist=== ====Convert Probelist to Fasta File==== *In Dropbox\GradZhangLab\CA12k_Oct2016\HumanBrain_V4\invitroCapture *Probelist: [[Media:padlockFile_0gap_HumanBrain_V4.txt]] **[[Media:CA12kOct2016V4_Probelist2Fasta.txt | CA12kOct2016V4_Probelist2Fasta.pl]] to generate ref file: CA12k_Oct2016_V4_H1H2.fa bowtie2-build CA12k_Oct2016_V4_H1H2.fa CA12k_Oct2016_V4_H1H2 ====Align==== bowtie2 --phred33 -x CA12k_Oct2016_V4_H1H2 -q Index1_S1_L001_R1_001.fastq > MC20161215_CA12kOct20161215_V4_T4_H1H2.sam 2> MC20161215_CA12kOct20161215_V4_T4_stderr.txt & 828460 reads; of these: 828460 (100.00%) were unpaired; of these: 621272 (74.99%) aligned 0 times 207179 (25.01%) aligned exactly 1 time 9 (0.00%) aligned >1 times 25.01% overall alignment rate bowtie2 --phred33 -x CA12k_Oct2016_V4_H1H2 -q Index2_S2_L001_R1_001.fastq > MC20161215_CA12kOct20161215_V4_SplintR_H1H2.sam 2> MC20161215_CA12kOct20161215_V4_SplintR_stderr.txt & 778774 reads; of these: 778774 (100.00%) were unpaired; of these: 699239 (89.79%) aligned 0 times 79525 (10.21%) aligned exactly 1 time 10 (0.00%) aligned >1 times 10.21% overall alignment rate bowtie2 --phred33 -x CA12k_Oct2016_V4_H1H2 -q Index3_S3_L001_R1_001.fastq > MC20161215_CA12kOct20161215_V4_Ampligase_H1H2.sam 2> MC20161215_CA12kOct20161215_V4_Ampligase_stderr.txt & 633888 reads; of these: 633888 (100.00%) were unpaired; of these: 342474 (54.03%) aligned 0 times 291410 (45.97%) aligned exactly 1 time 4 (0.00%) aligned >1 times 45.97% overall alignment rate ====Try Trimming to only good quality==== *Marginal improvement in alignment rate fastx_trimmer -Q33 -l 50 -i Index2_S2_L001_R1_001.fastq -o Index2_50bp.fastq bowtie2 --phred33 -x CA12k_Oct2016_V4_H1H2 -q Index2_50bp.fastq > MC20161215_CA12kOct20161215_V4_SplintR_50bp.sam 2> MC20161215_CA12kOct20161215_V4_SplintR_50bp_stderr.txt & 778774 reads; of these: 778774 (100.00%) were unpaired; of these: 559938 (71.90%) aligned 0 times 212096 (27.23%) aligned exactly 1 time 6740 (0.87%) aligned >1 times 28.10% overall alignment rate samtools view -bS MC20161215_CA12kOct20161215_V4_T4_H1H2.sam | samtools sort -o CA12kOct20161215_V4_T4_H1H2_sorted.bam samtools view -h -F 4 CA12kOct20161215_V4_T4_H1H2_sorted.bam > CA12kOct20161215_V4_T4_H1H2_sorted_filtered.sam samtools view -bS MC20161215_CA12kOct20161215_V4_SplintR_H1H2.sam | samtools sort -o CA12kOct20161215_V4_SplintR_H1H2_sorted.bam samtools view -h -F 4 CA12kOct20161215_V4_SplintR_H1H2_sorted.bam > CA12kOct20161215_V4_SplintR_H1H2_sorted_filtered.sam samtools view -bS MC20161215_CA12kOct20161215_V4_Ampligase_H1H2.sam | samtools sort -o CA12kOct20161215_V4_Ampligase_H1H2_sorted.bam samtools view -h -F 4 CA12kOct20161215_V4_Ampligase_H1H2_sorted.bam > CA12kOct20161215_V4_Ampligase_H1H2_sorted_filtered.sam ===Counting Reads for each Probe=== [[Media:CountReadsPer_Gene_Probe.txt | CountReadsPer_Gene_Probe.pl]] *CA12kOct20161215_V4_T4_H1H2_sorted_filtered.sam -> **CA12kOct20161215_V4_T4_H1H2_sorted_filtered_Genecounts.txt **CA12kOct20161215_V4_T4_H1H2_sorted_filtered_Probecounts.txt *CA12kOct20161215_V4_SplintR_H1H2_sorted_filtered.sam -> **CA12kOct20161215_V4_SplintR_H1H2_sorted_filtered_Genecounts.txt **CA12kOct20161215_V4_SplintR_H1H2_sorted_filtered_Probecounts.txt *CA12kOct20161215_V4_Ampligase_H1H2_sorted_filtered.sam -> **CA12kOct20161215_V4_Ampligase_H1H2_sorted_filtered_Genecounts.txt **CA12kOct20161215_V4_Ampligase_H1H2_sorted_filtered_Probecounts.txt <!-- ===Comparison=== [[File:20150712_With_vs_Without_Suppressorv2_Scatterplot.PNG|650px]] *Orange line is y=x *Blue line is trendline *The 4 genes suppressed by v2 design are labeled **Being left of y=x indicates suppressor oligos decreased the number of padlock probes capturing the gene/target ====Conclusions==== *Suppressorv2 is effective at decreasing capture counts of most captured genes *Good correlation between dT and Ran primer counts **6 genes did not appear in dT dataset but could be due to lower sequencing depth of that library (those genes should have poly-A tail) *NegCtrl correlates better with gDNA than RNA so probably a little DNA contamination in BA8 sample **Low enough that probably negligible ***eg. the cDNA Ran primer library was taken out of PCR before the NegCtrl curve started to go up [[Media:20150712_With_vs_Without_Suppressorv2.xlsx | Excel analysis]] -->
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information