Rui:DNAseq analysis on Hiseq120313: Difference between revisions

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* negative SNCs -> positive aliquot PCR
* negative SNCs -> positive aliquot PCR


====PCR primers====
{| {{table}} border=1
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|'''PCR-ID'''
| align="center" style="background:#f0f0f0;"|'''PCR-ID'''
Line 44: Line 45:
| align="center" style="background:#f0f0f0;"|'''refBase'''
| align="center" style="background:#f0f0f0;"|'''refBase'''
| align="center" style="background:#f0f0f0;"|'''B2B3(GT)'''
| align="center" style="background:#f0f0f0;"|'''B2B3(GT)'''
| align="center" style="background:#f0f0f0;"|'''positive aliquot'''
| align="center" style="background:#f0f0f0;"|'''Pf'''
| align="center" style="background:#f0f0f0;"|'''Pf'''
| align="center" style="background:#f0f0f0;"|'''Pr'''
| align="center" style="background:#f0f0f0;"|'''Pr'''
| align="center" style="background:#f0f0f0;"|'''PCR'''
| align="center" style="background:#f0f0f0;"|'''length'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''position'''
|-
|-
| SNC1||GLT25D2 (glycosyltransferase 25 domain containing 2)||exon||1||183907978||G||G=7/T=3||  CAGGGAGCCTTCATAGCTCA||  ACCTGAGTGACACGGAGACC||189bp||>chr1:183907885+183908073
| 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
|-
|-
| QC good||Fib||P30||B2||B3||B2_MDA||B3_MDA||||||||
| SNC3||PPP1R9A ||exon||7||94918006||G||A=3/G=6||B3_Indx82,B3_Indx95,B3_Indx96|| AGTGTGCAGCAGGTTTCTCA|| CCTCTCAGATATGCAGAATGCTC||223||>chr7:94917911+94918133
|-
|-
| PCR||1 band||1 band||||||||||||||||
| 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
|-
|-
| seq. ID||RL01_R||RL02||RL03||RL04||RL05||RL06||||||||
| 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)
|-
|-
| seq. length||350||160||160||160||160||70||||||||
| ||POU5F1P4 (in ASH1L)||exon, pseudogene||1||155403505||G||A=5/G=5||B2_Indx88,B2_Indx91,B2_Indx93,B3_Indx74,B3_Indx95||||||||
|-
|-
| blast||183907925 183908073||183907925 183908071||183907932 183908074||183907932 183908074||183907930 183908071||183907997 183908070||||||||
| SNC7||(in DAB1)||Ensemble psedogene||1||58694451||A||A=12/G=3||B2_Indx77,B2_Indx86,B3_Indx92|| CTGGGGTTCTTGGACCTTCT|| GATGCTCTGGGCCTCTGTC||178||>chr1:58694376+58694553
|-
|-
| identity||100%||100%||100%||100%||100%||100%||||||||
| 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
|-
|-
| SNC||G||G||G||G||G (T? C?)||N/A||||||||
| 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
|-
|-
| aliquot||||||B2_Indx80,B2_Indx93,B3_Indx93||||||||||||||
| 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|400px]] [[File:4.27.12_PCR8-14.jpg|400px]]


{| {{table}} border=1
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|'''PCR-ID'''
| align="center" style="background:#f0f0f0;"|'''PCR-ID'''
| align="center" style="background:#f0f0f0;"|'''Gene'''
| align="center" style="background:#f0f0f0;"|'''length'''
| align="center" style="background:#f0f0f0;"|'''Cat.'''
| align="center" style="background:#f0f0f0;"|'''PCR result'''
| align="center" style="background:#f0f0f0;"|'''chr'''
|-
| align="center" style="background:#f0f0f0;"|'''pos'''
| SNC1||189||good
| align="center" style="background:#f0f0f0;"|'''refBase'''
|-
| align="center" style="background:#f0f0f0;"|'''B2B3(GT)'''
| SNC2||191||weak
| align="center" style="background:#f0f0f0;"|'''Primers'''
|-
| align="center" style="background:#f0f0f0;"|'''PCR'''
| SNC3||223||good
|-
| SNC4||191||3 bands
|-
| SNC5-6||bad||3 bands
|-
| ||||
|-
|-
| SNC2||FLNB (actin binding protein 278)||exon||3||58104657||C||C=6/G=3||  TTTGGTACCCAAAGGTAAACTGA||191
| SNC7||178||good
|-
|-
| ||||||||||||||  CAACCCATCTGCTCTCCATT||>chr3:58104557+58104747
| SNC9||158||good
|-
|-
| failed, repeat||Fib||P30||B2||B3||B2_MDA||B3_MDA||||
| SNC10||369||good
|-
|-
| PCR||||||||||||||||
| SNC11-12||247||weak+smear
|-
|-
| seq. ID||RL09||RL10_R||RL11_R||RL12||RL13||RL14||||
| ||||
|-
|-
| seq. length||141||330||141||170||||70||||
| SNC14||176||good
|-
|-
| blast||183907930 183908070||58104610  58104747||58104607  58104747||183907950 183908060||||183907997 183908061||||
| SNC13||204||good
|-
|-
| identity||98%||100%||100%||94%||||||||
| SNC15||388||no
|-
|-
| SNC||SNC1? T?||C||C||SNC1? T?||no match||SNC1? N/A||||
| SNC16||204||>10 bands
|-
|-
| aliquot||||||||||||||B2_Indx80,B2_Indx82,B3_Indx92||
| SNC377||151||good
|}
|}


====Sequence results====
{| {{table}} border=1
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|'''PCR-ID'''
| align="center" style="background:#f0f0f0;"|'''PCR-ID'''
| align="center" style="background:#f0f0f0;"|'''length'''
| align="center" style="background:#f0f0f0;"|'''PCR result'''
| align="center" style="background:#f0f0f0;"|'''Gene'''
| align="center" style="background:#f0f0f0;"|'''Gene'''
| align="center" style="background:#f0f0f0;"|'''Cat.'''
| align="center" style="background:#f0f0f0;"|'''chr'''
| align="center" style="background:#f0f0f0;"|'''chr'''
| align="center" style="background:#f0f0f0;"|'''pos'''
| align="center" style="background:#f0f0f0;"|'''pos'''
| align="center" style="background:#f0f0f0;"|'''refBase'''
| align="center" style="background:#f0f0f0;"|'''refBase'''
| align="center" style="background:#f0f0f0;"|'''B2B3(GT)'''
| align="center" style="background:#f0f0f0;"|'''B2B3(GT)'''
| align="center" style="background:#f0f0f0;"|'''Primers'''
| align="center" style="background:#f0f0f0;"|'''Fib'''
| align="center" style="background:#f0f0f0;"|'''PCR'''
| align="center" style="background:#f0f0f0;"|'''P30'''
| align="center" style="background:#f0f0f0;"|'''B2'''
| align="center" style="background:#f0f0f0;"|'''B3'''
| align="center" style="background:#f0f0f0;"|'''B2_MDA'''
| align="center" style="background:#f0f0f0;"|'''B3_MDA'''
| align="center" style="background:#f0f0f0;"|'''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||||||||||||||
|-
|-
| SNC3||PPP1R9A ||exon||7||94918006||G||A=3/G=6|| AGTGTGCAGCAGGTTTCTCA||223
| SNC7||178||good||(in DAB1)||1||58694451||A||A=12/G=3||A||A||A||A||A||A||clear peak, confirm w/ aliquot
|-
|-
| |||||||||||||| CCTCTCAGATATGCAGAATGCTC||>chr7:94917911+94918133
| SNC9||158||good||||9||7598174||C||C=11/T=4||off target||C/T||chr21?||chr12?||chr2?||chr2?||repeat PCR (probmatic)
|-
|-
| QC good||Fib||P30||B2||B3||B2_MDA||B3_MDA||||
| SNC10||369||good||(in PLXNC1); repeat||12||94620564||T||C=3/T=13||||||||||||||repeats
|-
|-
| PCR||||||||||||||||
| SNC11-12||247||weak+smear||||9||7598190||A||A=10/G=5||||||||||||||
|-
|-
| seq. ID||RL17||RL18||RL19||RL20||RL21||RL22||||
| ||||||||9||7598203||C||C=12/T=5||||||||||||||
|-
|-
| seq. length||184||270||846, chimeric||1K, chimeric||198||1K||||
| SNC14||176||good||GOT2L2||10||93897290||A||A=11/G=3||A||A||A||A||A||A||clear peak, confirm w/ aliquot
|-
|-
| blast||94917950  94918133||94917950  94918132||94917950  94918133||94917951  94918133||94917949  94918133||||||
| 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
|-
|-
| identity||100%||100%||||||||||||
| SNC15||388||no||(in EP300)||22||41572232||G||A=3/G=8||G||SNC13?||G||G||G/A||no match||3 B2 aliquot
|-
|-
| SNC||G||G||G||G||G||chr1||||
| 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
|-
|-
| aliquot||||||||||||all B3||B3_Indx82,B3_Indx95,B3_Indx96||
| SNC377||151||good||SEZ6; repeat||17||27296926||T||C=3/T=10||T||T||mhits, T||off target||chr.15?||off target||repeats
|}
|}

Latest revision as of 23:15, 27 April 2012

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