Daniel:Notebook/GenomeMiner/2013-9-12

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Mock HL155 (Started 9/9/2013)

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Bowtie on Perfect Mock Sequence Data with Perfect Base Qualities

Based on the results from yesterday I'm seeing what kind of alignment bowtie2 does on perfect sequences with perfect base qualities. Yesterday's sequences were direct copies of the .fa file, but they used base qualities randomly picked from the original sequencing data. These base qualities are all I (an extremely high quality score for phred33).

Generated using MATLAB (MockHL155_Master, Switch 3)

scp to Genome Miner

scp V4S1_mockseq_perfseqbqual.fq djacobse@132.239.135.41:/media/LTS_15T/DEJ_LTS/SeqStore/130628_HL155/mockseq/

bowtie2

bowtie2 -x bbuild_v4s1 -U v4s1_mockseq_perfseqbqual.fq --phred33 -S mockseq_v4s1_perfseqbqual.sam

Alignment Results

2959000 reads; of these:
 2959000 (100.00%) were unpaired; of these:
   0 (0.00%) aligned 0 times
   2953000 (99.80%) aligned exactly 1 time
   6000 (0.20%) aligned >1 times
100.00% overall alignment rate

So it looks like even with indisputable base quality and sequences, about 0.2% of sequences are so close that bowtie2 thinks they can be several sequences.

Convert Sam to Bam and Sort

samtools view -bS mockseq_v4s1_perfseqbqual.sam | samtools sort - mockseq_v4s1_perfseqbqual_sort

Bowtie on Perfect Mock Sequence Data (Started 09/11/2013)

Using Matt's Perlscript: File:Imp count mismatch.txt

Error Rate: 0
Error Rate of Insertions: 0
Error Rate of Deletions: 0
Error Rate of Substitutions: 0
Counts: 2955000 perfect, 0 with error

So this is expected from the sequencing data with no errors. Interestingly, 4000 probes were not counted at all, meaning (most likely) that 4000 probes were of quality too low (they were filtered out in the calmd stage). 4000 is a suspicious number, being even, which could mean that 4 different probes were not very readable. It could also just be coincidence.