Rui:DNAseq analysis on Hiseq120313: Difference between revisions
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>RuiLiu m (→Validation) |
>RuiLiu |
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(2 intermediate revisions by the same user not shown) | |||
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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;"|''' | | align="center" style="background:#f0f0f0;"|'''length''' | ||
| align="center" style="background:#f0f0f0;"|'''''' | | align="center" style="background:#f0f0f0;"|'''position''' | ||
|- | |- | ||
| SNC1||GLT25D2 | | 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|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;"|''' | | align="center" style="background:#f0f0f0;"|'''length''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''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==== | |||
{| {{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;"|'''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;"|''' | | align="center" style="background:#f0f0f0;"|'''Fib''' | ||
| align="center" style="background:#f0f0f0;"|''' | | 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|||||||||||||| | |||
|- | |- | ||
| | | 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 | ||
|} | |} |
Latest revision as of 23:15, 27 April 2012
Hiseq_120313[edit]
Sample preparation[edit]
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
- An unique name for each library/index
- Excel or plain text for info/job files, tab-delimited plain text
- upload to triton (attn: space/line problems in nano format)
- tr "/r" "/n" < info.txt > info (convert to unix format)
- sed "s/73/xx/g" < 73.info > xx.info (convert to other index)
- qsub xx.job
- qstat | grep rul (check running status)
- qdel xxxx (kill job)
- 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 |