Average Read Coverage for 454 paired end read data

lisa1102 · 10-14-2011, 05:37 AM

Hi
I have just received some paired end read data from 454 flx sequencer. The coverage is 6X for three of my samples and 3X for one of my samples.
These samples are microbes with no reference strain.
Can anyone tell me if this coverage is the norm?
Thanks,
Lisa

MissDNA · 10-14-2011, 06:29 AM

Coverage depends on the size of your genomes. Do you have any idea of sizes?
Also, what kind of PE was it?

In our last 3kb PE run, we sequenced 2 bacteria in a 2 lane PTP. The 1st one had a genome around 4.5M and the coverage was 56X. The other one is larger than 7M and we got 16X coverage. We knew the 2nd library was not very good but decided to sequence anyway.

So if your organisms were small, I think 6x and 3x coverage is not very good.

pmiguel · 10-14-2011, 07:16 AM

Hi Lisa,
You will have to give us more to go on. Like how many and what type of regions were used on the 454 sequencer? Also what genome size are your microbes?

--
Phillip

vamosia · 10-18-2011, 08:10 AM

On my previous microbes projects, 20X seems to be the depth that works well with Newbler. More depth will typically break the contig more since the errors accumulate and less depth does not generate enough signal.

But this is not absolute and it depends on your genome.

lisa1102 · 10-18-2011, 08:25 AM

Hi
Thanks for your comments. This is the only information I have at present. The size of the genomes is 2.7MB for the 6X coverage and 1.5MB for the 3X coverage.
If people think that 20X is average then is anything I can do to improve the analyses or is it best to repeat the runs?

MissDNA · 10-18-2011, 08:28 AM

Don't you know how the PTP was divided: 2, 4, 8 or 16 regions? If you had a sample per region in a 2-lane PTP the coverage you got is way too low. How was the library prep?

lisa1102 · 10-18-2011, 08:36 AM

I don't have that information of the sample per region at the moment. I am waiting for the information. The only information I have on the library prep is from the protocol "Paired end lib prep method manual - 20kb and 8kb span"

MissDNA · 10-18-2011, 08:39 AM

I never worked with 8 and 20kb libraired. My experience is with 3kb.
My recomendation is you need to gather all the info: about quality of the library, number of reads generated, and how the PTP was divided before deciding what to do next.

lisa1102 · 10-18-2011, 08:40 AM

Yes Thanks for your comments. This will help me pose more specific questions.

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10-14-2011, 05:37 AM	#1
lisa1102 Junior Member Location: UK Join Date: Oct 2011 Posts: 4	Average Read Coverage for 454 paired end read data Hi I have just received some paired end read data from 454 flx sequencer. The coverage is 6X for three of my samples and 3X for one of my samples. These samples are microbes with no reference strain. Can anyone tell me if this coverage is the norm? Thanks, Lisa

10-14-2011, 06:29 AM	#2
MissDNA Senior Member Location: Brazil Join Date: Nov 2010 Posts: 146	Coverage depends on the size of your genomes. Do you have any idea of sizes? Also, what kind of PE was it? In our last 3kb PE run, we sequenced 2 bacteria in a 2 lane PTP. The 1st one had a genome around 4.5M and the coverage was 56X. The other one is larger than 7M and we got 16X coverage. We knew the 2nd library was not very good but decided to sequence anyway. So if your organisms were small, I think 6x and 3x coverage is not very good.

10-14-2011, 07:16 AM	#3
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,274	Hi Lisa, You will have to give us more to go on. Like how many and what type of regions were used on the 454 sequencer? Also what genome size are your microbes? -- Phillip

10-18-2011, 08:10 AM	#4
vamosia Member Location: New York Join Date: Mar 2009 Posts: 15	On my previous microbes projects, 20X seems to be the depth that works well with Newbler. More depth will typically break the contig more since the errors accumulate and less depth does not generate enough signal. But this is not absolute and it depends on your genome.

10-18-2011, 08:25 AM	#5
lisa1102 Junior Member Location: UK Join Date: Oct 2011 Posts: 4	Hi Thanks for your comments. This is the only information I have at present. The size of the genomes is 2.7MB for the 6X coverage and 1.5MB for the 3X coverage. If people think that 20X is average then is anything I can do to improve the analyses or is it best to repeat the runs?

10-18-2011, 08:28 AM	#6
MissDNA Senior Member Location: Brazil Join Date: Nov 2010 Posts: 146	Don't you know how the PTP was divided: 2, 4, 8 or 16 regions? If you had a sample per region in a 2-lane PTP the coverage you got is way too low. How was the library prep?

10-18-2011, 08:36 AM	#7
lisa1102 Junior Member Location: UK Join Date: Oct 2011 Posts: 4	I don't have that information of the sample per region at the moment. I am waiting for the information. The only information I have on the library prep is from the protocol "Paired end lib prep method manual - 20kb and 8kb span"

10-18-2011, 08:39 AM	#8
MissDNA Senior Member Location: Brazil Join Date: Nov 2010 Posts: 146	I never worked with 8 and 20kb libraired. My experience is with 3kb. My recomendation is you need to gather all the info: about quality of the library, number of reads generated, and how the PTP was divided before deciding what to do next.

10-18-2011, 08:40 AM	#9
lisa1102 Junior Member Location: UK Join Date: Oct 2011 Posts: 4	Yes Thanks for your comments. This will help me pose more specific questions.