BioMicroCenter:SPRI-Works: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
 
(6 intermediate revisions by the same user not shown)
Line 1: Line 1:
{{BioMicroCenter}}
{{BioMicroCenter}}
[[Image:DNA_prep_1.png|thumb|500px|right|Automation by the SPRIworks]]
Fragmented samples submitted to the BioMicro Center are processed using the Beckman Coulter SPRIworks. This system preforms the sample cleanup, a overhang, ligation and preliminary size selection needed for creating Illumina libraries. The system accepts all amounts of insert and has been extensively tested.


[[Image: BioMicro_SPRIworks.jpg|left|300px]][[Image:spri-works.jpg|right|200px]]
The SPRI-te Nucleic Acid Extractor is a fully-automated library preparation system for Illumina sequencing and supports all Illumina applications except for smallRNA-Seq. It performs all of the traditional Illumina protocol steps listed on the [[BioMicroCenter:Illumina Library Preparation|Illumina Library Preparation page]] with the exception of PCR enrichment. A total of 10 libraries may be prepared simultaneously in 5 hours.
The SPRI-te Nucleic Acid Extractor is a fully-automated library preparation system for Illumina sequencing and supports all Illumina applications except for smallRNA-Seq. It performs all of the traditional Illumina protocol steps listed on the [[BioMicroCenter:Illumina Library Preparation|Illumina Library Preparation page]] with the exception of PCR enrichment. A total of 10 libraries may be prepared simultaneously in 5 hours.


==How it Works==
== SPRIworks ==
{|
|- valign="top"
|
The SPRI-Works system  consists of three main components: the SPRI-TE Nucleic Acid Extractor, a method card that contains the liquid handling program, and a cartridge containing all of the reagents required to prepare a single library.
 
The platform utilizes AMPureXP DNA-binding magnetic beads to purify fragmented DNA and perform size selection, eliminating the need for column purification and gel-based size selection. After the adapter ligation and size selection steps are complete, samples are ready for PCR enrichment and clean-up to complete the library prep process. For more information on the SPRI-Works system please visit [http://spriworks.com www.spriworks.com].
 
|[[Image: BioMicro_SPRIworks.jpg|thumb|left|200px|SPRIworks]]
 
|}


The SPRI-Works system  consists of three main components: the SPRI-TE Nucleic Acid Extractor, a method card that contains the liquid handling program, and a cartridge containing all of the reagents required to prepare a single library.  
=== Size Selection ===
{|
|- valign="top"
|[[Image:spri-works.jpg|thumb|right|150px]]
|One of the more remarkable aspect of the SPRIbeads is their ability to provide size selection. SPRI beads are supplied in very high salt/PEG buffer that is needed for their binding. At low amounts of salt and PEG only large molecules are bound to the beads. As more salt is added, smaller fragmetns become bound to the DNA. This allows the "Double SPRI" size selection. A low amount of salt+beads is added to the mix and the high molecular weight material is discarded. Then additional beads (and salt) are added and a smaller size cut is taken with the beads while the low molecular weight material is discarded. On the SPRIworks, there are three size range options available, 200-400bp, 300-600bp, or no size selection. (These ranges apply to the inserts together with the partial adapters.)<br>
|}
 
=== Library Complexity ===
{| cellpadding="10"
|- valign="top"
|In order to test the performance of the SPRI-te, the same ChIP-seq sample underwent preparation for Illumina sequencing by both an experienced technician and on the SPRI-te. The numbers of unique sequencing reads and significant peaks were doubled in the SPRI-te samples when compared to the manual preparations.
|[[Image:DNA_prep_2.png|thumb|300px|right]]
|}


The platform utilizes AMPureXP DNA-binding magnetic beads to purify fragmented DNA and perform size selection, eliminating the need for column purification and gel-based size selection. There are three size range options available, 200-400bp, 300-600bp, or no size selection. (These ranges apply to the inserts together with the partial adapters.)<br>
=== Effect of Concentration ===
After the adapter ligation and size selection steps are complete, samples are ready for PCR enrichment and clean-up to complete the library prep process.
{| cellpadding="10"
|- valign=top


For more information on the SPRI-Works system please visit [http://spriworks.com www.spriworks.com].
|SPRIworks prepared samples have produced high-quality data even when sample input was below the manufacturer's recommendations. To test the dynamic range, we loaded varying quantities (200ng, 50, 12.5, 3.2, 0.8 and 0.2) of 50bp ladder from New England Biolabs on the SPRIworks to produce libraries. We then measured yield using qPCR and the Agilent BioAnalyzer. Results showed a consistent yield between 10% and 30% for the 200bp insert band consistent with the SPRI beads not being limiting.
|[[Image:DNA_prep_3.png|thumb|center|300px|Constructing Illumina libraries from 50bp ladder]]
|[[Image:DNA_prep_4.png|thumb|center|300px|Yield of library constructions as measured for the 200nt band]]
|- valign="top"
|[[Image:BMC_libraryComplexity.png|thumb|center|300px|Relationship between input material for SPRI and library complexity]]
|colspan="2"| Experience with the SPRIworks has enabled us to build up an understanding of the relationship between material submitted for the library and the complexity of the library. To the left is data from RNAseq libraries prepared for Sidi Chen of the Sharp lab. The linear correlation between number of molecules sequenced and the input is clearly visible. High complexity libraries required 1ng of input.
|}


==Sample Submission==
=== Sample Submission ===


Input sample concentrations on the SPRI-te are variable (we have successfully run samples as low as 0.3ng/uL). We suggest that you submit as much volume as possible since the first step of the protocol brings all sample volumes up to 100uL in water.  For optimal yield, libraries should be fragmented so that the average fragment size is approximately 60nt shorter than the chosen size range to allow for the addition of partial adapters.
We suggest that you submit as much volume as possible since the first step of the protocol brings all sample volumes up to 100uL in water.  For optimal yield, libraries should be fragmented so that the average fragment size is approximately 60nt shorter than the chosen size range to allow for the addition of partial adapters.


All samples submitted for '''BMC Fragmented DNA sample prep (SPRI-only)''' must be submitted with '''user supplied''' adapter mix. Adapter mixes should be pre-diluted and should have enough volume for 10uL of adapter per sample. For recommended dilutions for various sample concentrations, please see the chart below:
All samples submitted for '''BMC Fragmented DNA sample prep (SPRI-only)''' must be submitted with '''user supplied''' adapter mix. Adapter mixes should be pre-diluted and should have enough volume for 10uL of adapter per sample. For recommended dilutions for various sample concentrations, please see the chart below:

Latest revision as of 19:28, 19 June 2012

HOME -- SEQUENCING -- LIBRARY PREP -- HIGH-THROUGHPUT -- COMPUTING -- OTHER TECHNOLOGY
Automation by the SPRIworks

Fragmented samples submitted to the BioMicro Center are processed using the Beckman Coulter SPRIworks. This system preforms the sample cleanup, a overhang, ligation and preliminary size selection needed for creating Illumina libraries. The system accepts all amounts of insert and has been extensively tested.

The SPRI-te Nucleic Acid Extractor is a fully-automated library preparation system for Illumina sequencing and supports all Illumina applications except for smallRNA-Seq. It performs all of the traditional Illumina protocol steps listed on the Illumina Library Preparation page with the exception of PCR enrichment. A total of 10 libraries may be prepared simultaneously in 5 hours.

SPRIworks

The SPRI-Works system consists of three main components: the SPRI-TE Nucleic Acid Extractor, a method card that contains the liquid handling program, and a cartridge containing all of the reagents required to prepare a single library.

The platform utilizes AMPureXP DNA-binding magnetic beads to purify fragmented DNA and perform size selection, eliminating the need for column purification and gel-based size selection. After the adapter ligation and size selection steps are complete, samples are ready for PCR enrichment and clean-up to complete the library prep process. For more information on the SPRI-Works system please visit www.spriworks.com.

SPRIworks

Size Selection

 One of the more remarkable aspect of the SPRIbeads is their ability to provide size selection. SPRI beads are supplied in very high salt/PEG buffer that is needed for their binding. At low amounts of salt and PEG only large molecules are bound to the beads. As more salt is added, smaller fragmetns become bound to the DNA. This allows the "Double SPRI" size selection. A low amount of salt+beads is added to the mix and the high molecular weight material is discarded. Then additional beads (and salt) are added and a smaller size cut is taken with the beads while the low molecular weight material is discarded. On the SPRIworks, there are three size range options available, 200-400bp, 300-600bp, or no size selection. (These ranges apply to the inserts together with the partial adapters.)

Library Complexity

In order to test the performance of the SPRI-te, the same ChIP-seq sample underwent preparation for Illumina sequencing by both an experienced technician and on the SPRI-te. The numbers of unique sequencing reads and significant peaks were doubled in the SPRI-te samples when compared to the manual preparations.

Effect of Concentration

SPRIworks prepared samples have produced high-quality data even when sample input was below the manufacturer's recommendations. To test the dynamic range, we loaded varying quantities (200ng, 50, 12.5, 3.2, 0.8 and 0.2) of 50bp ladder from New England Biolabs on the SPRIworks to produce libraries. We then measured yield using qPCR and the Agilent BioAnalyzer. Results showed a consistent yield between 10% and 30% for the 200bp insert band consistent with the SPRI beads not being limiting.
Constructing Illumina libraries from 50bp ladder
Yield of library constructions as measured for the 200nt band
Relationship between input material for SPRI and library complexity
 Experience with the SPRIworks has enabled us to build up an understanding of the relationship between material submitted for the library and the complexity of the library. To the left is data from RNAseq libraries prepared for Sidi Chen of the Sharp lab. The linear correlation between number of molecules sequenced and the input is clearly visible. High complexity libraries required 1ng of input.

Sample Submission

We suggest that you submit as much volume as possible since the first step of the protocol brings all sample volumes up to 100uL in water. For optimal yield, libraries should be fragmented so that the average fragment size is approximately 60nt shorter than the chosen size range to allow for the addition of partial adapters.

All samples submitted for BMC Fragmented DNA sample prep (SPRI-only) must be submitted with user supplied adapter mix. Adapter mixes should be pre-diluted and should have enough volume for 10uL of adapter per sample. For recommended dilutions for various sample concentrations, please see the chart below:


Please note that SPRI-only samples will need to be re-submitted separately if you wish to sequence with us later on.

Sample Submission Forms

Retrieved from "https://openwetware.org/mediawiki/index.php?title=BioMicroCenter:SPRI-Works&oldid=608859"