Exon Sequencing of Human Chromosome X: I´m in serious trouble

frossit · 10-11-2011, 11:00 AM

Hello

My lab had discovered a locus in chromosome X that is involved with a kind of cortical malformation in a family with a pattern of X dominant inhiritance. We discovered this locus by linkage analysis. So we have performed exome enrichment of Ch X exome and second generation sequencing in a Illumina plataform. A paired end library was built (75 bp aproximatelly). Well ...there are a lot of duplicated genes and regions.... so bioinformatic analysis is not capable to say if region X is really X or Y ... because the homology is between 90 and 100% .... Thus my SNP calling is not reliable....
My supervisor told me to perform Sanger sequencing...but is almost impossible to pick primers in this region
There is other solution for my problem ?
Thanks

ulz_peter · 10-11-2011, 11:47 PM

Hi,

In case theduplicated regions are not too long, you could perform a long-range PCR (up to 25kb or so) and put the primers in regions with no duplication. This may serve as template for PCRs used for Sanger sequencing or may be directly sequenced (depending on the yield after LR-PCR).

Any other ideas? (Well you could do FlowSorting of Chromosomes, but that's not too easy)

frossit · 10-13-2011, 06:07 AM

Thanks Peter...
Unfortunately the region is long.... But your suggestion is very interesting...
I´m going to check if there is unique sequences between the repetitive regions... i can isolate its regions by long range PCR and use amplicons as a template for Sanger Sequencing...yes it´s possible !
Thank you very much
Best Regards
Fábio

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10-11-2011, 11:00 AM	#1
frossit Member Location: Brasil Join Date: Jun 2011 Posts: 12	Exon Sequencing of Human Chromosome X: I´m in serious trouble Hello My lab had discovered a locus in chromosome X that is involved with a kind of cortical malformation in a family with a pattern of X dominant inhiritance. We discovered this locus by linkage analysis. So we have performed exome enrichment of Ch X exome and second generation sequencing in a Illumina plataform. A paired end library was built (75 bp aproximatelly). Well ...there are a lot of duplicated genes and regions.... so bioinformatic analysis is not capable to say if region X is really X or Y ... because the homology is between 90 and 100% .... Thus my SNP calling is not reliable.... My supervisor told me to perform Sanger sequencing...but is almost impossible to pick primers in this region There is other solution for my problem ? Thanks

10-11-2011, 11:47 PM	#2
ulz_peter Senior Member Location: Graz, Austria Join Date: Feb 2010 Posts: 219	Hi, In case theduplicated regions are not too long, you could perform a long-range PCR (up to 25kb or so) and put the primers in regions with no duplication. This may serve as template for PCRs used for Sanger sequencing or may be directly sequenced (depending on the yield after LR-PCR). Any other ideas? (Well you could do FlowSorting of Chromosomes, but that's not too easy)

10-13-2011, 06:07 AM	#3
frossit Member Location: Brasil Join Date: Jun 2011 Posts: 12	Thanks Peter... Unfortunately the region is long.... But your suggestion is very interesting... I´m going to check if there is unique sequences between the repetitive regions... i can isolate its regions by long range PCR and use amplicons as a template for Sanger Sequencing...yes it´s possible ! Thank you very much Best Regards Fábio