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==Analysis of HL152: Representation Bias of CA12k Oligos (Corrected)== *I realized one of the parameters I have been using for bowtie2 ("-k 1") causes it to report the first good alignment but doesn't search for the best alignment *The other parameters that I changed from default (mismatch penalty, gap penalties) decreased the penalties from default allowing more alignments to be considered **The penalty for a single base gap is now equal to the penalty for single base mismatch while default a gap is about twice as high **This is okay for my purposes here because oligo synthesis introduces high rate of gaps but I still want to count those as aligned reads **I also want to consider as many reads as possible because I've shortened the reference to be very "specific"/defined (Not sure if this makes sense, I'll try to reword/explain better) Using the 50bp segments of the probe reference because it will create the fewest false alignments (aligning to full probe sequence resulted in ~10% more alignments): *Low alignment rate expected because did not use Illumina index so the reads included any junk reads without an index /home/kunzhang/softwares/bowtie2-latest/bowtie2 --phred64 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /home/mzcai/InSitu_HL152_130524_Analysis/CAprobes_50bp -q /home/mzcai/InSitu_HL152_130524_Analysis/s_2_1_unassigned.txt > /home/mzcai/InSitu_HL152_130524_Analysis/Readsalign2probes_CA.txt & mzcai@genome-miner:~/InSitu_HL152_130524_Analysis$ 29716630 reads; of these: 29716630 (100.00%) were unpaired; of these: 22676072 (76.31%) aligned 0 times 1719956 (5.79%) aligned exactly 1 time 5320602 (17.90%) aligned >1 times 23.69% overall alignment rate Counted up the reads for each probe using [[Media:Count_reads_from_bowtie2_SAM_quantifyOligo.txt|Count_reads_from_bowtie2_SAM_quantifyOligo.pl]]: CountofCAOligosv4.txt *Some variation is to be expected even for a perfectly uniform starting oligo pool because PCR is a stochastic process and each cycle will double some oligos and not others *Majority of probes have between 100-1000 reads **747 probes with >1000 reads **8 probes with <100 reads [[File:RankOrderofCA12kOligoCounts.JPG|border]] [[File:BinsofCA12kOligoCounts.JPG|border]] ===Using Default Bowtie2 Parameters=== *My previous idea of loosening the mismatch to consider more reads to find optimal alignment is no good **As shown below, using the default parameters results in a more Gaussian distribution (Poisson distribution for large numbers) that is expected **Also results in less alignments, which means instead of finding optimal alignments before, the lower penalties just allowed many poor reads align to specific probes, which resulted in extremely high read counts for them /home/kunzhang/softwares/bowtie2-latest/bowtie2 --phred64 -x /home/mzcai/InSitu_HL152_130524_Analysis/CAprobes_50bp -q /home/mzcai/InSitu_HL152_130524_Analysis/s_2_1_unassigned.txt > /home/mzcai/InSitu_HL152_130524_Analysis/Readsalign2probes_CA_default.txt & 29716630 reads; of these: 29716630 (100.00%) were unpaired; of these: 24520654 (82.51%) aligned 0 times 5156375 (17.35%) aligned exactly 1 time 39601 (0.13%) aligned >1 times 17.49% overall alignment rate Counted up reads: CountofCAOligosv5.txt *Average: 420.6 *Stdev: 162.8 (Gaussian) *Coefficient of Variation: 0.387 (Gaussian, stdev/mean) *Total number of aligned reads: 5,195,976 [[File:RankOrderofCA12kOligoCounts_default.JPG]]<br> [[File:BinsofCA12kOligoCounts_default.JPG]]<br>
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