Matt:LabNotes/2013-7-26: Difference between revisions
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**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 | **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 | **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): | 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): |
Revision as of 20:39, 26 July 2013
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 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
File:BinsofCA12kOligoCounts.JPG
Analysis of HL155: Representation Bias of Agi26k Oligos (Corrected)
- Using the same reasoning as for the CA12k Oligo alignment, redid alignment without "-k 1"
- Also separated 0gap and 20gap probes into two reference fasta files to prevent incorrect alignments
- As seen in the tables of [1], using a single fasta file resulted in many incorrect alignments
- This calls into the question the accuracy of reads/alignment
Fasta reference files (50bp long): Agi26k0gap.fa & Agi26k20gap.fa
Alignment of Agi26k oligo reads
- Low alignment rate for 0gap expected because ~2/3 Indx10 of the reads are Dan's probes
- Low alignment rate for 20gap expected because ~2/3 Indx12 of the reads are Dan's probes
0 gap probes
/home/kunzhang/softwares/bowtie2-latest/bowtie2 --phred64 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /home/mzcai/InSitu_HL155_130628_Analysis/Agi26k0gap -q /home/mzcai/InSitu_HL155_130628_Analysis/s_3_1_Indx10.txt > /home/mzcai/InSitu_HL155_130628_Analysis/Readsalign2probes_0gap.txt &
mzcai@genome-miner:~/InSitu_HL155_130628_Analysis$ 16368416 reads; of these: 16368416 (100.00%) were unpaired; of these: 15990987 (97.69%) aligned 0 times 127671 (0.78%) aligned exactly 1 time 249758 (1.53%) aligned >1 times 2.31% overall alignment rate
Counted up reads for each probe using Count_reads_from_bowtie2_SAM_quantifyOligo.pl: CountsofAgi26k_0gap.txt
- At least three reads for each probe
- 19 probes with >200 reads
- 154 probes with >100 reads
- 116 probes <10 reads
- 4 probes with <5 reads
File:RankOrderofAgi26k 0gapOligoCounts.JPG
File:BinsofAgi26k 0gapOligoCounts.JPG
20 gap probes
/home/kunzhang/softwares/bowtie2-latest/bowtie2 --phred64 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /home/mzcai/InSitu_HL155_130628_Analysis/Agi26k20gap -q /home/mzcai/InSitu_HL155_130628_Analysis/s_3_1_Indx12.txt > /home/mzcai/InSitu_HL155_130628_Analysis/Readsalign2probes_20gap.txt &
mzcai@genome-miner:~/InSitu_HL155_130628_Analysis$ 17308686 reads; of these: 17308686 (100.00%) were unpaired; of these: 17114997 (98.88%) aligned 0 times 63125 (0.36%) aligned exactly 1 time 130564 (0.75%) aligned >1 times 1.12% overall alignment rate
Counted up reads for each probe using Count_reads_from_bowtie2_SAM_quantifyOligo.pl: CountsofAgi26k_20gap.txt
- At least one read for each probe
- 18 probes with >100 reads
- 142 probes with >50 reads
- 311 probes with <5 reads