Exome sequencing

arvi8689 · 09-22-2011, 01:43 PM

Hey I am trying to sequence the melanoma exome to find somatic mutation involved in the melanoma. I am very new to this technique and I have some questions regarding this:

1)Generally how many exomes from different melanoma samples need to be done to get some satisfactory results?
2)What coverage is essential for such a study? Initially I was thinking of around 30X but some papers have used nearly 100X. Will I be okay with 30X coverage of the exome?

Your help is well appreciated.

hagir · 04-22-2012, 11:15 PM

hello every body, i'm a new member and hope that you help me
i want to perform an exome sequencing on oral cancer cases using illumina. i need you to help me with a good protocol for doing so
thanks

hagir · 04-22-2012, 11:15 PM

hello every body, i'm a new member and hope that you help me
i want to perform an exome sequencing on oral cancer cases using illumina. i need you to help me with a good protocol for doing so
thanks

AJERYC · 05-05-2012, 10:56 AM

30x is definitely not enough, since it means 30x averge, so you will get a lot of regions with very few sequences, that will make impossible for you to resolve heterozygous mutations. Go 50x or 100x.

ymc · 05-15-2012, 06:22 PM

Quote:

Originally Posted by AJERYC

30x is definitely not enough, since it means 30x averge, so you will get a lot of regions with very few sequences, that will make impossible for you to resolve heterozygous mutations. Go 50x or 100x.

How many x also depends on your enrichment kit. For example, according to the BGI paper, for 90% call sensitivity, Nimblegen SeqCap EZ v1 needs 50x, Agilent SureSelect All Exon needs 80x.

gensdei · 11-08-2012, 11:26 PM

Cell lines have the benefit of being a relatively pure population of clonal cells and thus represent a particular snapshot of cancers. Is there anybody who have seen a paper using exome-seq with 100x coverage based on cell line? If there is not, does it means that we don't need perform that deep sequencing due to its purity?

Thanks in advance!

AJERYC · 11-09-2012, 01:20 AM

[QUOTE=gensdei;88907]Cell lines have the benefit of being a relatively pure population of clonal cells and thus represent a particular snapshot of cancers. Is there anybody who have seen a paper using exome-seq with 100x coverage based on cell line? If there is not, does it means that we don't need perform that deep sequencing due to its purity?

Theoretically a cell line is homozygous and that is more easy to resolve with NGS so you could go 10-30x.

gensdei · 11-09-2012, 02:44 AM

[QUOTE=AJERYC;88920]

Quote:

Originally Posted by gensdei

Cell lines have the benefit of being a relatively pure population of clonal cells and thus represent a particular snapshot of cancers. Is there anybody who have seen a paper using exome-seq with 100x coverage based on cell line? If there is not, does it means that we don't need perform that deep sequencing due to its purity?

Theoretically a cell line is homozygous and that is more easy to resolve with NGS so you could go 10-30x.

To be more specific, exome-seq from cell line have no variant sites, e.g, SNP? Theoretically is it true? If so, you are right, no need for deep sequencing. What about exome-seq from cancer cell line? Did anybody see a paper on 100x exome-seq data from cancer cell line? Thanks.

mamons · 11-12-2012, 01:24 AM

Quote:

Originally Posted by hagir

hello every body, i'm a new member and hope that you help me
i want to perform an exome sequencing on oral cancer cases using illumina. i need you to help me with a good protocol for doing so
thanks

http://seqanswers.com/wiki/How-to/exome_analysis

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
PCR duplicate removal for whole genome sequencing vs. whole exome sequencing	cliff	Bioinformatics	1	09-27-2011 07:29 AM
Exome sequencing	Dan_cham	SOLiD	1	04-11-2011 11:18 PM
Exome Sequencing	Shruti Madhiwalla	Bioinformatics	13	01-03-2011 07:00 PM
Hands-on ngs workshop - human exome sequencing and microbial whole genome sequencing	vikram	Events / Conferences	0	12-08-2010 08:36 PM
Exome Sequencing	mollusc	Illumina/Solexa	1	12-01-2010 02:49 AM

09-22-2011, 01:43 PM	#1
arvi8689 Member Location: Boston Join Date: Sep 2011 Posts: 10	Exome sequencing Hey I am trying to sequence the melanoma exome to find somatic mutation involved in the melanoma. I am very new to this technique and I have some questions regarding this: 1)Generally how many exomes from different melanoma samples need to be done to get some satisfactory results? 2)What coverage is essential for such a study? Initially I was thinking of around 30X but some papers have used nearly 100X. Will I be okay with 30X coverage of the exome? Your help is well appreciated.

04-22-2012, 11:15 PM	#3
hagir Junior Member Location: malaysia Join Date: Apr 2012 Posts: 3	exome sequencing hello every body, i'm a new member and hope that you help me i want to perform an exome sequencing on oral cancer cases using illumina. i need you to help me with a good protocol for doing so thanks

05-05-2012, 10:56 AM	#4
AJERYC Member Location: Spain Join Date: Jan 2012 Posts: 22	30x is definitely not enough, since it means 30x averge, so you will get a lot of regions with very few sequences, that will make impossible for you to resolve heterozygous mutations. Go 50x or 100x.

11-08-2012, 11:26 PM	#6
gensdei Junior Member Location: seoul Join Date: Jul 2012 Posts: 2	Cell lines have the benefit of being a relatively pure population of clonal cells and thus represent a particular snapshot of cancers. Is there anybody who have seen a paper using exome-seq with 100x coverage based on cell line? If there is not, does it means that we don't need perform that deep sequencing due to its purity? Thanks in advance!

11-09-2012, 01:20 AM	#7
AJERYC Member Location: Spain Join Date: Jan 2012 Posts: 22	[QUOTE=gensdei;88907]Cell lines have the benefit of being a relatively pure population of clonal cells and thus represent a particular snapshot of cancers. Is there anybody who have seen a paper using exome-seq with 100x coverage based on cell line? If there is not, does it means that we don't need perform that deep sequencing due to its purity? Theoretically a cell line is homozygous and that is more easy to resolve with NGS so you could go 10-30x.