Matt:LabNotes/2013-8-27

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Revision as of 01:23, 31 August 2013 by >Mzcai (→‎Checking Error Rate for V4S1)
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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%