Matt:LabNotes/2013-7-26: Difference between revisions
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==Analysis of HL155: Representation Bias of Agi26k Oligos (Corrected)== | ==Analysis of HL155: Representation Bias of Agi26k Oligos (Corrected)== | ||
*WAS USING THE WRONG END OF PROBES AS REFERENCE WHICH WAS WHY ALIGNMENT RATE WAS < 3% (see hidden comments for that analysis) | |||
**Create new fasta file with [[File:Probelist_to_fasta_revcomp.txt|Probelist_to_fasta_revcomp.pl]] | |||
*Try both full reference (Agi26k_corrected.fa) and split between 0gap and 20gap (Agi26k0gap_corrected.fa & Agi26k20gap_corrected.fa) | |||
===Alignment of Agi26k oligo reads to full reference=== | |||
*~33% alignment rate for 0gap expected because ~2/3 Indx10 of the reads are Dan's probes | |||
*~33% alignment rate for 20gap expected because ~2/3 Indx12 of the reads are Dan's probes | |||
*Using the same reasoning as for the CA12k Oligo alignment, redid alignment without "-k 1" | |||
<!--*Using the same reasoning as for the CA12k Oligo alignment, redid alignment without "-k 1" | <!--*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 | *Also separated 0gap and 20gap probes into two reference fasta files to prevent incorrect alignments |
Revision as of 00:20, 27 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)
- WAS USING THE WRONG END OF PROBES AS REFERENCE WHICH WAS WHY ALIGNMENT RATE WAS < 3% (see hidden comments for that analysis)
- Create new fasta file with Probelist_to_fasta_revcomp.pl
- Try both full reference (Agi26k_corrected.fa) and split between 0gap and 20gap (Agi26k0gap_corrected.fa & Agi26k20gap_corrected.fa)
Alignment of Agi26k oligo reads to full reference
- ~33% alignment rate for 0gap expected because ~2/3 Indx10 of the reads are Dan's probes
- ~33% alignment rate for 20gap expected because ~2/3 Indx12 of the reads are Dan's probes
- Using the same reasoning as for the CA12k Oligo alignment, redid alignment without "-k 1"