Matt:LabNotes/2013-7-26: Difference between revisions
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Counted up reads for each probe using [[Media:Count_reads_from_bowtie2_SAM_quantifyOligo.txt|Count_reads_from_bowtie2_SAM_quantifyOligo.pl]]: CountsofAgi26k_0gap.txt | Counted up reads for each probe using [[Media:Count_reads_from_bowtie2_SAM_quantifyOligo.txt|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 | |||
*4 probes with <5 reads | |||
[[File:RankOrderofAgi26k_0gapOligoCounts.JPG|border]] | [[File:RankOrderofAgi26k_0gapOligoCounts.JPG|border]] | ||
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Counted up reads for each probe using [[Media:Count_reads_from_bowtie2_SAM_quantifyOligo.txt|Count_reads_from_bowtie2_SAM_quantifyOligo.pl]]: CountsofAgi26k_20gap.txt | Counted up reads for each probe using [[Media:Count_reads_from_bowtie2_SAM_quantifyOligo.txt|Count_reads_from_bowtie2_SAM_quantifyOligo.pl]]: CountsofAgi26k_20gap.txt | ||
*At least one read for each probe | *At least one read for each probe | ||
*18 probes with >100 reads | |||
*142 probes with >50 reads | |||
[[File:RankOrderofAgi26k_20gapOligoCounts.JPG|border]] | [[File:RankOrderofAgi26k_20gapOligoCounts.JPG|border]] | ||
[[File:BinsofAgi26k_20gapOligoCounts.JPG|border]] | [[File:BinsofAgi26k_20gapOligoCounts.JPG|border]] |
Revision as of 18:21, 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
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
- 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