Matt:LabNotes/2013-8-27
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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
' | start | end | 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%
Error Rate of Insertions: 0.0739448051948052 = 7.4%
Error Rate of Deletions: 0.368181818181818 = 36.8%
Error Rate of Substitutions: 0.00373376623376623 = 0.37%
- Realized Mistake in MYcroArray Reference Sequences: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-8-30