Matt:LabNotes/2013-7-26

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Revision as of 17:58, 26 July 2013 by >Mzcai (Created page with "==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 g...")
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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

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

File:RankOrderofAgi26k 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

File:RankOrderofAgi26k 20gapOligoCounts.JPG

Counted up reads for each probe using Count_reads_from_bowtie2_SAM_quantifyOligo.pl: CountsofAgi26k_20gap.txt