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==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 *For Regression Analysis with RNA-Seq, FISSEQ, and BeadArray (PGP1f) see: [[Matt:LabNotes/2015-4-8#Regression_Analysis]] ===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 --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=== [[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 **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==== <!--TFM only */media/LTS_33T/BL_LTS33T/hNuc/Rizi_Bulk_Controls **RL-BA8-sec9-t-N701-15May14_S1/Counts_Gene (bulk tissue) **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==== *[[Media:RCommands_CA12kNov2014_V4CaptureAnalysis.txt | Some R commands used]] *Dropbox/GradZhangLab/CA12k_Nov2014/V4_CaptureAnalysis/V4CaptureAnalysis.RData **All_GeneCounts is data.frame with all data ====Results==== *Since 66 (1.8%) 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===== *Regression of only genes where DARTFISH does not target masked regions <-----------> Including genes targeting masked regions [[File:BA8_logDARTFISH_vs_logRNASeqBulkN_masked_regression.jpeg|450px]] [[File:BA8_logDARTFISH_vs_logRNASeqBulkN_regression.jpeg|450px]] =====DARTFISH vs Bulk Tissue===== *Regression of only genes where DARTFISH does not target masked regions <-----------> Including genes targeting masked regions [[File:BA8_logDARTFISH_vs_logRNASeqBulkT_masked_regression.jpeg|450px]] [[File:BA8_logDARTFISH_vs_logRNASeqBulkT_regression.jpeg|450px]] *Regression of only genes where DARTFISH does not target masked regions **Excluded high and low expression genes based on RNA-Seq (-1 < log_FPKM < 8) [[File:BA8_logDARTFISH_vs_logRNASeqBulkT_masked_medExpressGenes_regression.jpeg|450px]] ===Conclusion=== *in situ DARTFISH gene counts correlates better with bulk tissue RNA-Seq *While removing genes with probes that target masked regions is conservative and certainly correct, it barely improves correlation with RNA-Seq **Below are histograms of invitro gene counts, LEFT: all probes for gene considered, MIDDLE: only genes with no probes targeting masked region, RIGHT: partially masked - all 240 genes included but probes that target masked region not counted [[File:Hist_CA12kNov2014_V4_ReadsPerGene_allprobes.jpeg | 350px]] [[File:Hist_CA12kNov2014_V4_ReadsPerGene_masked.jpeg | 350px]] [[File:Hist_CA12kNov2014_V4_ReadsPerGene_partmasked.jpeg | 350px]] *Need to design [[Media:CA12kNov2014_V4_SoftMaskedProbes.txt | 66 suppressor oligos]] to completely suppress (ordered probes flagged with a 1 in last column of file) **Maybe only 12 probes with >1,000 count to suppress the probes that have astronomical in situ counts ***18 probes with > 500, 22 probes with > 200, 32 probes with > 100 ***Use histogram of reads per probe (with softmaskedprobes removed) to decide [[File:Hist_CA12kNov2014_V4_ReadsPerProbe_masked.jpeg | 450px]] [[File:Hist_CA12kNov2014_V4_ReadsPerProbe_maskedtargets.jpeg | 450px]] *Ordered 39 probes **13 probes with >850 count **26 probes (with 50 < count < 850 and >~30% lowercase) + (probes with 11 < count < 50 and >~30% lowercase and show up in DARTFISH data)
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