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Old 08-22-2014, 07:55 AM   #1
xiefanfang@gmail.com
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Question Illumina sequencing for multiplexed RRBS

Our lab is interested in doing multiplexed reduced representative bisulfite sequencing (RRBS) on mouse DNA. This is the first time for our lab to use this technique and we plan to follow the steps described in the paper “Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling”. Because this paper was published in 2012, I don't know if techniques have been improved since then. Specifically, I have the following questions regarding sample preparation and sequencing.

1. For sample preparation, can I use TruSeq DNA PCR-Free kit for multiplexing?

2. Are the following steps necessary for sequencing as described in the paper? We are going to do sequencing in an outside institute, and we only have 6-12 samples and want to use 1-2 lanes of a Hiseq2000 or Hiseq2500 flow cell. I am afraid that having these specialized sequencing steps will affect the other lanes in the same flow cell.

“The MspI recognition cut site (C^CGG) creates fragments that will make the first three bases of every read non-random. This would result in high apparent cluster density, poor DNA cluster localization, and significant data loss during sequencing on the Illumina HiSeq 2000. To improve performance of these samples and increase coverage obtained, we used a method referred to as 'dark sequencing' in which imaging and cluster localization were delayed until the fourth cycle of sequencing chemistry, beyond the extent of bias from the MspI cut site (Figure S3 in Additional file1).
To do this, we loaded a HiSeq 2000 with a custom recipe file co-developed with Illumina plus extra reagents to support primer re-hybridization. The custom recipe created a new initial 'template read' in which the first three biased bases were incorporated without imaging, followed by four cycles that were incorporated, imaged, and used by the sequencer for cluster localization. Next, the recipe removed the newly synthesized strand using NaOH and a buffer wash, re-hybridized fresh sequencing primer to the sample, and began read 1 data collection as usual from the first base but using the pre-existing cluster map or 'template' generated by the template read. HiSeq Control Software (HCS) provided by Illumina prevented cluster intensity files from the template read to enter downstream analysis.
As all custom chemistry steps were defined by the recipe, this workflow required very little additional hands-on time compared to a standard HiSeq run setup. The template read took approximately 6 h and consumed seven cycles of sequencing reagents prior to the start of data collection. Additional reagents to support re-hybridization after the template read were loaded at the beginning of the run alongside other read 1 and index read sequencing reagents. The following positions differed from the standard setup for an indexed single read run: Pos 16, 3 ml Read 1 Sequencing primer; Pos 18, 5 ml 0.1 N NaOH, Pos 19, 6 ml Illumina wash buffer.”

If you have updated protocols for multiplexed RRBS, please send me a copy. I also look forward to your reply. Thank you very much!!
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Old 08-22-2014, 02:45 PM   #2
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The University of Oregon Sequencing Facility sequences a lot of RAD-Seq libraries, which also start with a cut site sequence. The HiSeq is sensitive to low complexity in the first bases, but there are a few easier steps you can take. 1) Have a control lane that is high complexity (phiX or shotgun). 2) Don't overload. 3) Spike in some high-complexity material into your lane (10%). Steps 1 & 2 should be enough, but 3 can help.
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Old 08-23-2014, 01:26 AM   #3
nucacidhunter
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Quote:
Because this paper was published in 2012, I don't know if techniques have been improved since then.
Following-up papers citing that one should reveal if there has been any improvement.

Quote:
1. For sample preparation, can I use TruSeq DNA PCR-Free kit for multiplexing?
Adapters for TruSeq LT PCR-Free and Nano kits both are methylated, so they can be used for RRBS. However, adapters for dual indexed versions are not methylated and should be avoided.

Quote:
2. Are the following steps necessary for sequencing as described in the paper? We are going to do sequencing in an outside institute, and we only have 6-12 samples and want to use 1-2 lanes of a Hiseq2000 or Hiseq2500 flow cell. I am afraid that having these specialized sequencing steps will affect the other lanes in the same flow cell.
Sequencing RRBS libraries with initial dark cycles is the most cost effective work around to avoid low complexity issues, but it requires an updated custom recipe similar to that one which Illumina normally would provide. In this case the best option would be HiSeq RAPID run in one flow cell (2 lanes). Custom recipes are applied to whole flow cell so high output flow cells will not be cost effective for your sequencing needs. The issue will be finding a service provider willing to do this. I guess only providers that are using this custom recipe regularly would be willing to do it just for two lanes.

If dark cycle sequencing is not possible, work around mentioned by SNPsaurus is most likely the only other option.
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Old 08-25-2014, 07:59 AM   #4
xiefanfang@gmail.com
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Quote:
Originally Posted by SNPsaurus View Post
The University of Oregon Sequencing Facility sequences a lot of RAD-Seq libraries, which also start with a cut site sequence. The HiSeq is sensitive to low complexity in the first bases, but there are a few easier steps you can take. 1) Have a control lane that is high complexity (phiX or shotgun). 2) Don't overload. 3) Spike in some high-complexity material into your lane (10%). Steps 1 & 2 should be enough, but 3 can help.
Thank you! I did some research on Phix control and it is a good idea to have a control lane and spike the sample libraries. Do you have a working protocol regarding this method? Also, because my samples are bisulfite converted, do they require a higher concentration spike-in, such as 40% or higher? Do you know how to calculate phasing and prephaisng values? Thank you very much!
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Old 08-28-2014, 02:12 PM   #5
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I held off responding because of this:
http://res.illumina.com/documents/pr...oISq2tWbgNo6cK

The HiSeq now does an excellent job of low-diversity sequencing, it looks like!
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