Rui:DNAseq analysis on Hiseq120313

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Hiseq_120313[edit]

Sample preparation[edit]

[1]

Flow cell[edit]

  • s1-2: RL_B2_23s_Mar8.2012
  • s5-6: RL_B3_24s_Mar8.2012
  • s7-8: RL_Fib-Bulk_Mar8.2012

Mapping[edit]

  • fastq
Genome-miner: /media/SeqStore2/120315_HiSeq/Genome
Triton: /projects/zhang-lab/seqStore/120313_HiSeq/Genome
  • prepare to submit jobs in triton
info file: File:B2 Indx73.info.txt
job file: File:B2 Indx73.job.txt
  1. An unique name for each library/index
  2. Excel or plain text for info/job files, tab-delimited plain text
  3. upload to triton (attn: space/line problems in nano format)
  4. tr "/r" "/n" < info.txt > info (convert to unix format)
  5. sed "s/73/xx/g" < 73.info > xx.info (convert to other index)
  6. qsub xx.job
  7. qstat | grep rul (check running status)
  8. qdel xxxx (kill job)
  9. triton contact info: Jim Hayes <jhayes@sdsc.edu> [2]
  • following analysis
[3]


Validation[edit]

  • multiple bands / unknown sequence in PCR results -> confirm primers on gDNA
  • negative SNCs -> positive aliquot PCR

PCR primers[edit]

PCR-ID Gene Cat. chr pos refBase B2B3(GT) positive aliquot Pf Pr length position
SNC1 GLT25D2 exon 1 183907978 G G=7/T=3 B2_Indx80,B2_Indx93,B3_Indx93 CAGGGAGCCTTCATAGCTCA ACCTGAGTGACACGGAGACC 189 >chr1:183907885+183908073
SNC2 FLNB exon 3 58104657 C C=6/G=3 B2_Indx80,B2_Indx82,B3_Indx92 TTTGGTACCCAAAGGTAAACTGA CAACCCATCTGCTCTCCATT 191 >chr3:58104557+58104747
SNC3 PPP1R9A exon 7 94918006 G A=3/G=6 B3_Indx82,B3_Indx95,B3_Indx96 AGTGTGCAGCAGGTTTCTCA CCTCTCAGATATGCAGAATGCTC 223 >chr7:94917911+94918133
SNC4 POU5F1P3 (in CLEC4A) exon, pseudogene 12 8286643 C C=13/G=6 B2_Indx81,B2_Indx83,B2_Indx87,B2_Indx93,B3_Indx83,B3_Indx88 GAGACCCAACAGCCTCAAAA AGTGAGAGGCAACCTGGAGA 191 >chr12:8286540+8286730
SNC5-6 POU5F1P4 (in ASH1L) exon, pseudogene 1 155403537 G C=5/G=6 B2_Indx81,B2_Indx88,B2_Indx91,B2_Indx93,B3_Indx74 AACAATTTGCCAAGCTCCTG TGCAAGAGGGTTTCTGCTTT bad chr1; chr8; chr6 (X5)
POU5F1P4 (in ASH1L) exon, pseudogene 1 155403505 G A=5/G=5 B2_Indx88,B2_Indx91,B2_Indx93,B3_Indx74,B3_Indx95
SNC7 (in DAB1) Ensemble psedogene 1 58694451 A A=12/G=3 B2_Indx77,B2_Indx86,B3_Indx92 CTGGGGTTCTTGGACCTTCT GATGCTCTGGGCCTCTGTC 178 >chr1:58694376+58694553
SNC9 Ensemble psedogene 9 7598174 C C=11/T=4 B2_Indx73,B2_Indx81,B2_Indx84,B3_Indx73 CACTGGCAGCAAGTCAATCT GCCATCCAACCACTCAGTCT 158 >chr9:7598095+7598252 158bp
SNC10 (in PLXNC1) intron; repeats 12 94620564 T C=3/T=13 B2_Indx91,B3_Indx75,B3_Indx96 CCAGGTAAAGTGACATTTTTGG AGGACCAGGCAGTCACAAAC 369 >chr12:94620480+94620848 369bp
SNC11-12 Ensemble psedogene 9 7598190 A A=10/G=5 B2_Indx73,B2_Indx79,B2_Indx81,B2_Indx84,B3_Indx73 GGGTTCCTGCTTTCACAAAA TTGCTGAAGACCACATGCTT 247 >chr9:7598026+7598272 247bp
Ensemble psedogene 9 7598203 C C=12/T=5 B2_Indx73,B2_Indx79,B2_Indx81,B2_Indx84,B3_Indx73
SNC14 GOT2L2 exon, pseudogene 10 93897290 A A=11/G=3 B2_Indx74,B2_Indx86,B3_Indx84 CCAAAAGCTCAACAGCAACA TCCACTCCTCATGTTCCACA 176 >chr10:93897208+93897383 176bp
SNC13 EIF4A1P8 (in CPEB3) exon, pseudogene 1 173111263 C C=7/T=3 B3_Indx82,B3_Indx84,B3_Indx92 TCACCCAGGGTTGGTTCTAC TGGGAGTCACTGAAGCCTTT 204 >chr1:173111163+173111366 204bp
SNC15 (in EP300) intron 22 41572232 G A=3/G=8 B2_Indx79,B2_Indx88,B2_Indx91 GAGGATCAGGATCAACAATGG GTGCTCAGCCTGCTGTACCT 388 >chr22:41571936-41572323 388bp
SNC16 UPF3A exon 13 115047559 C C=5/T=3 B2_Indx74,B2_Indx78,B2_Indx86 GTTCCGCCCTCTCTTCCTC CCTTTTGAGCTCCTTGTCCA 204 >chr13:115047437-115047640 204bp
SNC377 SEZ6 exon; repeats 17 27296926 T C=3/T=10 B2_Indx74,B2_Indx91,B3_Indx76 AGGCAGCAGGAAGAAGTCTG CAGCAGGAAAGACTGGTTGG 151 >chr17:27296875-27297025 151bp
  • PCR product from primers on P30 gDNA

File:4.27.12 PCR1-7.jpg File:4.27.12 PCR8-14.jpg

PCR-ID length PCR result
SNC1 189 good
SNC2 191 weak
SNC3 223 good
SNC4 191 3 bands
SNC5-6 bad 3 bands
SNC7 178 good
SNC9 158 good
SNC10 369 good
SNC11-12 247 weak+smear
SNC14 176 good
SNC13 204 good
SNC15 388 no
SNC16 204 >10 bands
SNC377 151 good

Sequence results[edit]

PCR-ID length PCR result Gene chr pos refBase B2B3(GT) Fib P30 B2 B3 B2_MDA B3_MDA Notes
SNC1 189 good GLT25D2 1 183907978 G G=7/T=3 G G G G G (T? C?) N/A confirm w/ aliquot
SNC2 191 weak FLNB 3 58104657 C C=6/G=3 SNC1? T? C [330bp] C SNC1? T? no match SNC1? N/A repeat PCR (probmatic)
SNC3 223 good PPP1R9A 7 94918006 G A=3/G=6 G G [270bp] G G G chr1
SNC4 191 3 bands POU5F1P3 (in CLEC4A) 12 8286643 C C=13/G=6 C/G G/A/C G A C: chr12; G:chr8/1
SNC5-6 bad 3 bands POU5F1P4 (in ASH1L) 1 155403537 G C=5/G=6
POU5F1P4 (in ASH1L) 1 155403505 G A=5/G=5
SNC7 178 good (in DAB1) 1 58694451 A A=12/G=3 A A A A A A clear peak, confirm w/ aliquot
SNC9 158 good 9 7598174 C C=11/T=4 off target C/T chr21? chr12? chr2? chr2? repeat PCR (probmatic)
SNC10 369 good (in PLXNC1); repeat 12 94620564 T C=3/T=13 repeats
SNC11-12 247 weak+smear 9 7598190 A A=10/G=5
9 7598203 C C=12/T=5
SNC14 176 good GOT2L2 10 93897290 A A=11/G=3 A A A A A A clear peak, confirm w/ aliquot
SNC13 204 good EIF4A1P8 (in CPEB3) 1 173111263 C C=7/T=3 C C C C C/T/G C chr17,12,13 etc.; mosaic
SNC15 388 no (in EP300) 22 41572232 G A=3/G=8 G SNC13? G G G/A no match 3 B2 aliquot
SNC16 204 >10 bands UPF3A 13 115047559 C C=5/T=3 chr. 12?-off chr. 17?-off chr. 17: T chr. 17? chr13-C; chr17-C, T
SNC377 151 good SEZ6; repeat 17 27296926 T C=3/T=10 T T mhits, T off target chr.15? off target repeats