Matt:LabNotes/2015-3-19: Difference between revisions
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==CA12k_Nov2014_V4 in vitro Capture Sequencing Analysis== | ==CA12k_Nov2014_V4 in vitro Capture Sequencing Analysis== | ||
*With in vitro Capture data, DARTFISH gene counts can be normalized and compared to other methods such as RNA-Seq | |||
CA12kNov2014_Probelist2Fasta.pl | ===Mapping MiSeq reads to Oligo Sequences=== | ||
====Convert Probelist to Fasta File==== | |||
*Probelist: [[Media:OutputFile_0gap_contig_final.full_info.V4.txt]] | |||
[[Media:CA12kNov2014_Probelist2Fasta.txt | CA12kNov2014_Probelist2Fasta.pl]] | |||
bowtie2-build CA12k_Nov2014_V4_H1H2.fa CA12k_Nov2014_V4_H1H2 | bowtie2-build CA12k_Nov2014_V4_H1H2.fa CA12k_Nov2014_V4_H1H2 | ||
bowtie2 --phred33 -x CA12k_Nov2014_V4_H1H2 -q MC-20150121_CA12kNov2014_V4gDNA-1.R1.fastq > CA12kNov2014_V4gDNA_R1_H1H2.sam 2> CA12kNov2014_V4gDNA_stderr.txt & | bowtie2 --phred33 -x CA12k_Nov2014_V4_H1H2 -q MC-20150121_CA12kNov2014_V4gDNA-1.R1.fastq > CA12kNov2014_V4gDNA_R1_H1H2.sam 2> CA12kNov2014_V4gDNA_stderr.txt & | ||
2573875 reads; of these: | 2573875 reads; of these: | ||
2573875 (100.00%) were unpaired; of these: | 2573875 (100.00%) were unpaired; of these: | ||
Line 12: | Line 16: | ||
72 (0.00%) aligned >1 times | 72 (0.00%) aligned >1 times | ||
95.50% overall alignment rate | 95.50% overall alignment rate | ||
bowtie2 --phred33 -x CA12k_Nov2014_V4_H1H2 -q MC-20150121_CA12kNov2014_V4cDNA-2.R1.fastq > CA12kNov2014_V4cDNA_R1_H1H2.sam 2> CA12kNov2014_V4cDNA_stderr.txt & | bowtie2 --phred33 -x CA12k_Nov2014_V4_H1H2 -q MC-20150121_CA12kNov2014_V4cDNA-2.R1.fastq > CA12kNov2014_V4cDNA_R1_H1H2.sam 2> CA12kNov2014_V4cDNA_stderr.txt & | ||
2425688 reads; of these: | 2425688 reads; of these: | ||
2425688 (100.00%) were unpaired; of these: | 2425688 (100.00%) were unpaired; of these: | ||
Line 26: | Line 30: | ||
samtools view -h -F 4 CA12kNov2014_V4cDNA_R1_H1H2_sorted.bam > CA12kNov2014_V4cDNA_R1_H1H2_sorted_filtered.sam | samtools view -h -F 4 CA12kNov2014_V4cDNA_R1_H1H2_sorted.bam > CA12kNov2014_V4cDNA_R1_H1H2_sorted_filtered.sam | ||
CountReadsPer_Gene_Probe.pl | ===Count # of Reads Aligned to each Probe=== | ||
[[Media:CountReadsPer_Gene_Probe.txt | CountReadsPer_Gene_Probe.pl]] | |||
*Verified with grep -c 'Gene' CA12kNov2014_V4gDNA_R1_H1H2_sorted_filtered.sam and grep -c 'Probe' CA12kNov2014_V4gDNA_R1_H1H2_sorted_filtered.sam | *Verified with grep -c 'Gene' CA12kNov2014_V4gDNA_R1_H1H2_sorted_filtered.sam and grep -c 'Probe' CA12kNov2014_V4gDNA_R1_H1H2_sorted_filtered.sam | ||
**Differences in counts are accounted for by strings in header lines | **Differences in counts are accounted for by strings in header lines | ||
===Compare with Blue's BA8 bulk RNA-seq Gene Counts=== | ===Compare with Blue's BA8 bulk RNA-seq Gene Counts=== | ||
====RNA-Seq Data on Genome-miner==== | |||
<!--TFM only | |||
*/media/LTS_33T/BL_LTS33T/hNuc/Rizi_Bulk_Controls | */media/LTS_33T/BL_LTS33T/hNuc/Rizi_Bulk_Controls | ||
**RL-BA8-sec9-t-N701-15May14_S1/Counts_Gene (bulk tissue) | **RL-BA8-sec9-t-N701-15May14_S1/Counts_Gene (bulk tissue) | ||
**RL-BA8-sec9-n-N702-15May14_S2/Counts_Gene (bulk sorted neuronal nuclei) | **RL-BA8-sec9-n-N702-15May14_S2/Counts_Gene (bulk sorted neuronal nuclei)--> | ||
*mapped_genes.fpkm_tracking.txt | |||
**/media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/RL-BA8-sec9-n-N702-15May14_S2_mapped | |||
***neuronal nuclei | |||
**/media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/RL-BA8-sec9-t-N701-15May14_S1_mapped | |||
***bulk tissue | |||
====Analysis in R==== | |||
====Results==== | |||
*Since 40 (1%) probes have target regions that are highly repetitive (softmasked in reference genome; lowercase), they can have very high false in vitro capture counts and are removed from analysis | |||
=====DARTFISH vs Sorted Neuronal Nuclei==== | |||
[[File:BA8_logDARTFISH_vs_logRNASeqBulkN_masked_regression.jpeg]] | |||
*Including probes targeting masked regions | |||
[[File:BA8_logDARTFISH_vs_logRNASeqBulkN_regression.jpeg]] | |||
====DARTFISH vs Bulk Tissue==== | |||
[[File:BA8_logDARTFISH_vs_logRNASeqBulkT_masked_regression.jpeg]] | |||
*Including probes targeting masked regions | |||
[[File:BA8_logDARTFISH_vs_logRNASeqBulkT_regression.jpeg]] |
Revision as of 01:01, 24 March 2015
CA12k_Nov2014_V4 in vitro Capture Sequencing Analysis
- With in vitro Capture data, DARTFISH gene counts can be normalized and compared to other methods such as RNA-Seq
Mapping MiSeq reads to Oligo Sequences
Convert Probelist to Fasta File
CA12kNov2014_Probelist2Fasta.pl
bowtie2-build CA12k_Nov2014_V4_H1H2.fa CA12k_Nov2014_V4_H1H2
bowtie2 --phred33 -x CA12k_Nov2014_V4_H1H2 -q MC-20150121_CA12kNov2014_V4gDNA-1.R1.fastq > CA12kNov2014_V4gDNA_R1_H1H2.sam 2> CA12kNov2014_V4gDNA_stderr.txt & 2573875 reads; of these: 2573875 (100.00%) were unpaired; of these: 115745 (4.50%) aligned 0 times 2458058 (95.50%) aligned exactly 1 time 72 (0.00%) aligned >1 times 95.50% overall alignment rate bowtie2 --phred33 -x CA12k_Nov2014_V4_H1H2 -q MC-20150121_CA12kNov2014_V4cDNA-2.R1.fastq > CA12kNov2014_V4cDNA_R1_H1H2.sam 2> CA12kNov2014_V4cDNA_stderr.txt & 2425688 reads; of these: 2425688 (100.00%) were unpaired; of these: 110744 (4.57%) aligned 0 times 2314903 (95.43%) aligned exactly 1 time 41 (0.00%) aligned >1 times 95.43% overall alignment rate
samtools view -bS CA12kNov2014_V4gDNA_R1_H1H2.sam | samtools sort - CA12kNov2014_V4gDNA_R1_H1H2_sorted samtools view -h -F 4 CA12kNov2014_V4gDNA_R1_H1H2_sorted.bam > CA12kNov2014_V4gDNA_R1_H1H2_sorted_filtered.sam
samtools view -bS CA12kNov2014_V4cDNA_R1_H1H2.sam | samtools sort - CA12kNov2014_V4cDNA_R1_H1H2_sorted samtools view -h -F 4 CA12kNov2014_V4cDNA_R1_H1H2_sorted.bam > CA12kNov2014_V4cDNA_R1_H1H2_sorted_filtered.sam
Count # of Reads Aligned to each Probe
- Verified with grep -c 'Gene' CA12kNov2014_V4gDNA_R1_H1H2_sorted_filtered.sam and grep -c 'Probe' CA12kNov2014_V4gDNA_R1_H1H2_sorted_filtered.sam
- Differences in counts are accounted for by strings in header lines
Compare with Blue's BA8 bulk RNA-seq Gene Counts
RNA-Seq Data on Genome-miner
- mapped_genes.fpkm_tracking.txt
- /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/RL-BA8-sec9-n-N702-15May14_S2_mapped
- neuronal nuclei
- /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/RL-BA8-sec9-t-N701-15May14_S1_mapped
- bulk tissue
- /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/RL-BA8-sec9-n-N702-15May14_S2_mapped
Analysis in R
Results
- Since 40 (1%) probes have target regions that are highly repetitive (softmasked in reference genome; lowercase), they can have very high false in vitro capture counts and are removed from analysis
=DARTFISH vs Sorted Neuronal Nuclei
File:BA8 logDARTFISH vs logRNASeqBulkN masked regression.jpeg
- Including probes targeting masked regions
File:BA8 logDARTFISH vs logRNASeqBulkN regression.jpeg
DARTFISH vs Bulk Tissue
File:BA8 logDARTFISH vs logRNASeqBulkT masked regression.jpeg
- Including probes targeting masked regions