Matt:LabNotes/2015-3-19: Difference between revisions

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   samtools view -bS CA12kNov2014_V4cDNA_R1_H1H2.sam | samtools sort - CA12kNov2014_V4cDNA_R1_H1H2_sorted
   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
   samtools view -h -F 4 CA12kNov2014_V4cDNA_R1_H1H2_sorted.bam > CA12kNov2014_V4cDNA_R1_H1H2_sorted_filtered.sam
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===
*/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)

Revision as of 18:22, 20 March 2015

CA12k_Nov2014_V4 in vitro Capture Sequencing Analysis

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

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

  • /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)