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===Bowtie2 and Novoalign of Dan's MYcroArray Probes=== *Fastq Reads: /media/LTS_15T/DEJ_LTS/SeqStore/130628_HL155/fastq/ **V6S1:Lane 3 Index 11 **V4S1:Lane 2 Index 13 **V6S2:Lane 3 Index 10 **V4S2:Lane 3 Index 12 **V6S3:Lane 3 Index 11 **V4S3:Lane 2 Index 13 **V6S4:Lane 3 Index 10 **V4S4:Lane 3 Index 12 *Fasta Reference: /media/LTS_15T/DEJ_LTS/SeqStore/130628_HL155/probeseq/Probelist_all.fa {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''start''' | align="center" style="background:#f0f0f0;"|'''end''' | align="center" style="background:#f0f0f0;"|'''x2''' |- | V6S1||1||16559||33118 |- | V4S1||16560||19518||39036 |- | V6S2||19519||35918||71836 |- | V4S2||35919||39518||79036 |- | V6S3||39519||52518||105036 |- | V4S3||52519||58368||116736 |- | V6S4||58369||71682||143364 |- | V4S4||71683||77669||155338 |} ====Plan==== *Shorten reference file to 50bp with Editfasta.pl *Separate probe reference into 8 sets head -33118 Probelist_50bp.fa > Probelist_V6S1.fa head -39036 Probelist_50bp.fa | tail -5918 > Probelist_V4S1.fa head -71836 Probelist_50bp.fa | tail -32800 > Probelist_V6S2.fa head -79036 Probelist_50bp.fa | tail -7200 > Probelist_V4S2.fa head -105036 Probelist_50bp.fa | tail -26000 > Probelist_V6S3.fa head -116736 Probelist_50bp.fa | tail -11700 > Probelist_V4S3.fa head -143364 Probelist_50bp.fa | tail -26628 > Probelist_V6S4.fa head -155338 Probelist_50bp.fa | tail -11974 > Probelist_V4S4.fa *Build novoalign indexes for each /home/kunzhang/softwares/Novocraft/novocraft/novoindex V6S1.ndx Probelist_V6S1.fa /home/kunzhang/softwares/Novocraft/novocraft/novoindex V4S1.ndx Probelist_V4S1.fa /home/kunzhang/softwares/Novocraft/novocraft/novoindex V6S2.ndx Probelist_V6S2.fa /home/kunzhang/softwares/Novocraft/novocraft/novoindex V4S2.ndx Probelist_V4S2.fa /home/kunzhang/softwares/Novocraft/novocraft/novoindex V6S3.ndx Probelist_V6S3.fa /home/kunzhang/softwares/Novocraft/novocraft/novoindex V4S3.ndx Probelist_V4S3.fa /home/kunzhang/softwares/Novocraft/novocraft/novoindex V6S4.ndx Probelist_V6S4.fa /home/kunzhang/softwares/Novocraft/novocraft/novoindex V4S4.ndx Probelist_V4S4.fa *Do end-to-end alignment with novoalign /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d V6S1.ndx -f s_3_1_Indx11.txt -F ILMFQ -r ALL -o SAM -o FULLNW > V6S1_novoalign.sam 2> V6S1_stderr.txt /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d V4S1.ndx -f s_2_1_Indx13.txt -F ILMFQ -r ALL -o SAM -o FULLNW > V4S1_novoalign.sam 2> V4S1_stderr.txt /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d V6S2.ndx -f s_3_1_Indx10.txt -F ILMFQ -r ALL -o SAM -o FULLNW > V6S2_novoalign.sam 2> V6S2_stderr.txt /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d V4S2.ndx -f s_3_1_Indx12.txt -F ILMFQ -r ALL -o SAM -o FULLNW > V4S2_novoalign.sam 2> V4S2_stderr.txt /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d V6S3.ndx -f s_3_1_Indx11.txt -F ILMFQ -r ALL -o SAM -o FULLNW > V6S3_novoalign.sam 2> V6S3_stderr.txt /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d V4S3.ndx -f s_2_1_Indx13.txt -F ILMFQ -r ALL -o SAM -o FULLNW > V4S3_novoalign.sam 2> V4S3_stderr.txt /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d V6S4.ndx -f s_3_1_Indx10.txt -F ILMFQ -r ALL -o SAM -o FULLNW > V6S4_novoalign.sam 2> V6S4_stderr.txt /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d V4S4.ndx -f s_3_1_Indx12.txt -F ILMFQ -r ALL -o SAM -o FULLNW > V4S4_novoalign.sam 2> V4S4_stderr.txt ====Checking Error Rate for V4S1==== # Read Sequences: 11450664 # Aligned: 1061 # Unique Alignment: 1061 # Gapped Alignment: 1061 # Quality Filter: 23301 # Homopolymer Filter: 40 # Elapsed Time: 10198.104 (sec.) # CPU Time: 164.2 (min.) # Done at Fri Aug 30 14:34:36 2013 samtools view -bS V4S1_novoalign.sam | samtools sort - V4S1_novoalign_sorted samtools view -h -F 4 -q 70 V4S1_novoalign_sorted.bam > V4S1_novoalign_sorted_filtered.sam samtools calmd -eS V4S1_novoalign_sorted_filtered.sam Probelist_V4S1.fa > V4S1_novoalign_sf=.sam (After the first 30 bases, ALL WRONG (insertions)) perl Count_mismatches_from_novoalignSAMquantifyOligo.pl -> CountMismatches_calmd_V4S1_novoalign.txt Error Rate: 0.44586038961039 = 44.6%<br> Error Rate of Insertions: 0.0739448051948052 = 7.4%<br> Error Rate of Deletions: 0.368181818181818 = 36.8%<br> Error Rate of Substitutions: 0.00373376623376623 = 0.37%<br> *Realized Mistake in MYcroArray Reference Sequences: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-8-30
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