Daniel:Notebook/GenomeMiner/2013-8-27: Difference between revisions
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bowtie2 -x hrcp_probesequences -U s_2_1_Indx12.txt,s_2_1_Indx13.txt,s_3_1_Indx11.txt,s_3_1_Indx10.txt, | bowtie2 -x hrcp_probesequences -U s_2_1_Indx12.txt,s_2_1_Indx13.txt,s_3_1_Indx11.txt,s_3_1_Indx10.txt, | ||
s_3_1_Indx12.txt,s_2_1_Indx11.txt --phred64 -S hrcp_slimindex_samout_p64.sam | s_3_1_Indx12.txt,s_2_1_Indx11.txt --phred64 -S hrcp_slimindex_samout_p64.sam | ||
'''Results''' | |||
55964494 reads; of these: | |||
55964494 (100.00%) were unpaired; of these: | |||
35040039 (62.61%) aligned 0 times | |||
19308407 (34.50%) aligned exactly 1 time | |||
1616048 (2.89%) aligned >1 times | |||
37.39% overall alignment rate | |||
If you look above, you'll see that ~20000 less reads aligned exactly one time and about 1500 less reads aligned greater than one time, meaning about 21500 reads were aligned that shouldn't have been when I was using the full index. As such, I'm going to continue the pipeline but using the sam files for the fewer indexes. | |||
'''Sam > Bam and Sort Bam''' | |||
samtools view -bS hrcp_slimindex_samout_p64.sam | samtools sort - hrcp_sorted_hl155 & | |||
'''Filter and Convert back to Sam''' | |||
samtools view -h -F 4 -q 8 hrcp_sorted_hl155.bam > hrcp_sorted_filtered_slimindex_hl155.sam & | |||
'''Replace Matched pairs with Equals''' | |||
samtools calmd -eS hrcp_sorted_filtered_slimindex_hl155.sam ../probeseq/Probelist_all.fa > hrcp_sortfilt_slimindex_eq.sam |
Latest revision as of 17:27, 28 August 2013
HL155[edit]
Running Bowtie2 with Phred 64[edit]
Running bowtie2 again with phred 64 to start the pipeline again. Tried last week but it didn't go so well. I got an error. So I'm running the program again.
Bowtie2 with Phred 64
bowtie2 -x hrcp_probesequences -U s_2_1_Indx12.txt,s_2_1_Indx53.txt,s_2_1_Indx54.txt,s_2_1_Indx04.txt,s_2_1_Indx03.txt,s_2_1_Indx05.txt,s_2_1_Indx02.txt, s_2_1_Indx13.txt,s_2_1_Indx01.txt,s_2_1_Indx55.txt,s_2_1_Indx49.txt,s_3_1_Indx11.txt,s_3_1_Indx10.txt,s_2_1_Indx56.txt, s_2_1_Indx52.txt,s_3_1_Indx12.txt,s_2_1_Indx11.txt,s_2_1_Indx50.txt --phred64 -S hrcp_fullindex_samout_p64.out
Results:
71613792 reads; of these: 71613792 (100.00%) were unpaired; of these: 50674903 (70.76%) aligned 0 times 19321427 (26.98%) aligned exactly 1 time 1617462 (2.26%) aligned >1 times 29.24% overall alignment rate
Sam > Bam and Sort Bam
samtools view -bS hrcp_fullindex_samout_p64.sam | samtools sort - hrcp_sorted_hl155 &
Filter and Convert back to Sam
samtools view -h -F 4 -q 8 hrcp_sorted_hl155.bam > hrcp_sorted_filtered_hl155.sam &
Replace Matched pairs with Equals
samtools calmd -eS hrcp_sorted_filtered_hl155.sam ../probeseq/Probelist_all.fa > hrcp_sortfilt_eq.sam
So apparently I don't need to use all the indexes. I just used indexes 10-13, which means I can skip everything but those. Using all the indexes might screw up some of the statistics, mostly if someones sequence was close enough to have an alignment (a bad one, most likely).
Phred 64, Shaved Indexing[edit]
Bowtie2 with Phred 64 and Fewer Indexes
bowtie2 -x hrcp_probesequences -U s_2_1_Indx12.txt,s_2_1_Indx13.txt,s_3_1_Indx11.txt,s_3_1_Indx10.txt, s_3_1_Indx12.txt,s_2_1_Indx11.txt --phred64 -S hrcp_slimindex_samout_p64.sam
Results
55964494 reads; of these: 55964494 (100.00%) were unpaired; of these: 35040039 (62.61%) aligned 0 times 19308407 (34.50%) aligned exactly 1 time 1616048 (2.89%) aligned >1 times 37.39% overall alignment rate
If you look above, you'll see that ~20000 less reads aligned exactly one time and about 1500 less reads aligned greater than one time, meaning about 21500 reads were aligned that shouldn't have been when I was using the full index. As such, I'm going to continue the pipeline but using the sam files for the fewer indexes.
Sam > Bam and Sort Bam
samtools view -bS hrcp_slimindex_samout_p64.sam | samtools sort - hrcp_sorted_hl155 &
Filter and Convert back to Sam
samtools view -h -F 4 -q 8 hrcp_sorted_hl155.bam > hrcp_sorted_filtered_slimindex_hl155.sam &
Replace Matched pairs with Equals
samtools calmd -eS hrcp_sorted_filtered_slimindex_hl155.sam ../probeseq/Probelist_all.fa > hrcp_sortfilt_slimindex_eq.sam