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

File:RankOrderofAgi26k 20gapOligoCounts.JPG

File:BinsofAgi26k 20gapOligoCounts.JPG