BioMicroCenter:Illumina Library Preparation

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{{BioMicroCenter}}
{{BioMicroCenter}}
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[[Image:Presentationbest.jpg|right|400px]]
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[[Image:BMC_samplePrep_pipes.png|thumb|right|400px|Sample Preparation available in BioMicro]]
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Generating high-quality data on the Illumina sequencing platform requires high-quality libraries. The BMC currently offers library preparation services for genomic DNA and RNA-seq. Prior to sequencing, all samples must pass the BioMicro Center’s [[BioMicroCenter:Sequencing_Quality_Control|Sequencing Quality Control]] process, which verifies selection of inserts of a desired size and correct ligation of Illumina adapters.<br>Questions can be directed to [[BioMicroCenter:People|Michael Gravina and Ryan Sinapius]].
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Generating high-quality data on the Illumina sequencing platform requires high-quality libraries. The BMC currently offers library preparation services for a variety of starting materials. Prior to sequencing, all samples must pass the BioMicro Center’s [[BioMicroCenter:Sequencing_Quality_Control|Sequencing Quality Control]] process, which verifies selection of inserts of a desired size and correct ligation of Illumina adapters.<br>Sample Prep questions can be directed to [[BioMicroCenter:People|Shmulik Motola]].
==Sample Prep Services==
==Sample Prep Services==
=== DNA ===
=== DNA ===
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There are three options when submitting DNA for library prep:
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There are two options when submitting DNA for library prep depending on the type of sample submitted.
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'''[[BioMicroCenter:DNA Sample Preparation|BMC Fragmented DNA sample prep (SPRI and enrichment)]]''' - Samples will be pre-QC'd by [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]],  run on the [[BioMicroCenter:SPRI-Works|SPRI-works]] system using BioMicro Center adapters (with size-selection at the user's option), enriched using BioMicro Center PCR primers (including molecular barcodes for multiplexing), final-QC'd, and either submitted for Illumina sequencing or returned to the user for their own sequencing.
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'''[[BioMicroCenter:DNA Sample Preparation|Fragmented DNA]]''' - Samples will be pre-QC'd by [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]],  run on the [[BioMicroCenter:SPRI-Works|SPRI-works]] system using BioMicro Center adapters (with size-selection at the user's option), enriched using BioMicro Center PCR primers (including molecular barcodes for multiplexing), final-QC'd, and either submitted for Illumina sequencing or returned to the user for their own sequencing. Users may also chose an a la carte service where only SPRI is preformed.  
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'''[[BioMicroCenter:SPRI-Works|BMC Fragmented DNA sample prep (SPRI-only)]]''' - Samples will be run on the [[BioMicroCenter:SPRI-Works|SPRI-works]] system using '''user-supplied''' adapter mix and then returned to the user for their own enrichment. When choosing this option, please note that samples will need to be re-submitted if you plan to sequence with us later on.
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'''[[BioMicroCenter:Nextera|BMC Intact DNA sample prep (NEXTERA)]]''' - Samples will be pre-QC'd by Nanodrop, processed using the Epicentre Nextera kit, enriched using Nextera PCR primers (including molecular barcodes if requested), final-QC'd on the [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]] and either submitted for Illumina sequencing or returned to the user for their own sequencing.  At least 50ng of DNA is required for Nextera. Samples for Nextera cannot be pre-QC'ed on the [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]] because the large fragments will clog the microchannels.
'''[[BioMicroCenter:Nextera|BMC Intact DNA sample prep (NEXTERA)]]''' - Samples will be pre-QC'd by Nanodrop, processed using the Epicentre Nextera kit, enriched using Nextera PCR primers (including molecular barcodes if requested), final-QC'd on the [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]] and either submitted for Illumina sequencing or returned to the user for their own sequencing.  At least 50ng of DNA is required for Nextera. Samples for Nextera cannot be pre-QC'ed on the [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]] because the large fragments will clog the microchannels.
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=== RNA ===
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'''[[BioMicroCenter:PippinPrep|Size Selection]]''' - For samples requiring tight size selection, amplified libraries can be size selected using the [[BioMicroCenter:PippinPrep|PippinPrep]] prior to sequencing.
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'''mRNA-seq''' - Samples will be pre-QC'd on the [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]], poly-A purified and converted to cDNA using the Illumina Tru-Seq protocol, run on the SPRI-works system using BioMicro Center adapters, enriched using BioMicro Center PCR primers, and submitted for Illumina Sequencing.
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=== [[BioMicroCenter:RNAseq|RNA]] ===
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=== Chromatin ===
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There are several options when submitting RNA for library prep. These options are largely dictated by the source of the RNA, the quality of the RNA and the amount of RNA available.In addition, strand specific information is available for some methods and tight size selection is available for all methods.
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ChIP-Seq: Sonicated chromatin will undergo IP on the IPstar. RNase A, Proteinase K, Phenol Chloroform extraction and purification will be performed off of the instrument and submitted for QC on the Bioanalyzer. These ChIP samples may be picked up for qPCR or sent directly to SPRI for library construction and sequencing (options are listed on submission forms). Users will be contacted if ChIP samples fail QC. These samples will not be submitted to SPRI-works but will be charged for the IP.  
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'''mRNA-seq''' - Samples will be pre-QC'd on the [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]], poly-A purified and converted to cDNA using the Illumina Tru-Seq protocol, run on the [[BioMicroCenter:SPRI-Works|SPRI-works]] system using BioMicro Center adapters, enriched using BioMicro Center PCR primers, and submitted for Illumina sequencing.
 +
'''Strand Specific mRNA-seq''' - Samples will be pre-QC'd on the [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]], poly-A purified and converted to cDNA using the dUTP 2nd strand marking protocol outlined in J. Levin et al 2010, run on the [[BioMicroCenter:SPRI-Works|SPRI-works]] system using BioMicro Center adapters, enriched using BioMicro Center PCR primers, and submitted for Illumina sequencing.
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'''[[BioMicroCenter:Forms#Downloads|Sample Submission Forms]]'''<br><br><br>
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'''Low Input mRNA-seq''' - Samples will be pre-QC'd on the [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]], poly-A purified and converted to cDNA using the Ultra Low Input mRNAseq Kit from Clontech, run on the [[BioMicroCenter:SPRI-Works|SPRI-works]] system using BioMicro Center adapters, enriched using BioMicro Center PCR primers, and submitted for Illumina sequencing.
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==Submission Requirements==
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=== [[BioMicroCenter:ChIP||Chromatin]] ===
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'''DNA'''– DNA samples must be submitted in either water or TE. Although the [[BioMicroCenter:SPRI-Works|SPRIworks]] robot is not highly sensitive to the amount of DNA input, care must be taken to submit an [[BioMicroCenter:SPRI-Works|appropriate amount of '''adapter''' to ensure efficient ligation]]. If you elect the Nextera kit instead of the SPRIworks, we will require 50ng of DNA input.
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'''ChIP-Seq''': Sonicated chromatin will undergo IP on the IPstar. RNase A, Proteinase K, Phenol:Chloroform extraction and purification will be performed off of the instrument and submitted for QC on the [[BioMicroCenter:2100BioAnalyzer|BioAnalyzer]]. These ChIP samples may be picked up for qPCR or sent directly to [[BioMicroCenter:SPRI-Works|SPRI]] for library construction and sequencing (options are listed on submission forms). Users will be contacted if ChIP samples fail QC. These samples will not be submitted to SPRI-works but will be charged for the IP.
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== Barcoding ==
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Our '''[[BioMicroCenter:DNA Sample Preparation|BMC Fragmented DNA sample prep (SPRI and enrichment)]]''' service includes [[BioMicroCenter:Multiplex|molecular barcoding]] to allow sequencing of multiple samples per lane. Nextera DNA prep and RNAseq-prep may include barcoding at the user's option.
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'''RNA''' – Total RNA samples must be submitted in water or TE. The quantity of total RNA needs to be between 0.1-4ug with a RIN above 9.0.
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==Submission==
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Unless otherwise instructed, the BMC reserves 50% of submitted DNA in case of failures during sample prep. If you would like us to utilize the full sample, please indicate it in the notes section. <br><br>
 +
'''DNA'''– DNA samples must be submitted in either water or TE. Although the [[BioMicroCenter:SPRI-Works|SPRIworks]] robot is not highly sensitive to the amount of DNA input, care must be taken to submit an [[BioMicroCenter:SPRI-Works|appropriate amount of '''adapter''' to ensure efficient ligation]]. If you select the Nextera kit instead of the SPRIworks, we will require 50ng of DNA input.
 +
 +
'''RNA''' – Total RNA samples must be submitted in water or TE. The quantity of total RNA needs to be between 0.1-4ug for Illumina TruSeq, 1ug-4ug for strand specific RNAseq, >100pg for Low Input RNAseq.
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'''Chromatin''' – Sonicated chromatin must be submitted at 5 million cells per 200uL volume. Antibodies must be submitted at the same time(at least 3ug of antibody per IP). We have done months of testing and highly recommend using the following sonication conditions:
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[[BioMicroCenter:ChIP|'''Chromatin''']] – Sonicated chromatin must be submitted at 5-7 million cells per 200uL volume. Antibodies must be submitted at the same time(at least 3ug of antibody per IP). Chromatin should be prepared on the SAME DAY as the ChIP whenever possible. Full details about isolation methods can be found on the [[BioMicroCenter:ChIP|chromatin IP page]].
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1. Resuspend fixed cells in 10mL of [[LB1|LB1]] <br>
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'''[[BioMicroCenter:Forms#Downloads|Sample Submission Forms]]'''<br><br><br>
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2. Rock cells at 4 degrees for 10 minutes <br>
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3. Pellet cells at 4 degrees for 5 minutes at 1350g <br>
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4. Remove supernatant and resuspend cells in 10mL of [[LB2|LB2]] <br>
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5. Rock cells at 4 degrees for 10 minutes <br>
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6. Pellet cells at 4 degrees for 5 minutes at 1350g <br>
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7. Remove supernatant and resuspend cells in a volume of [[LB3|LB3]] where there is 5 million cells/200uL <br>
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'''**Example:''' Resuspend 25 million cells in 1mL of [[LB3|LB3]]'''**'''
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8. Sonicate cells <br>
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9. Pellet debris at 4 degrees for 10 minutes at 20,000g <br>
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10. Submit lysate for ChIP
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== QC ==
== QC ==
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Good quality control is crucial for optimizing the number of reads and quality of data produced by the sequencers. For more information on QC methods and protocols please visit the [[BioMicroCenter:Sequencing_Quality_Control|Sequencing Quality Control]] page. <br><br><br>
Good quality control is crucial for optimizing the number of reads and quality of data produced by the sequencers. For more information on QC methods and protocols please visit the [[BioMicroCenter:Sequencing_Quality_Control|Sequencing Quality Control]] page. <br><br><br>
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==Sample Prep Workflow==
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==Workflow==
[[Image: Libprep3.jpg|right|450px]]
[[Image: Libprep3.jpg|right|450px]]
* Fragmentation - All samples must be fragmented to the point where the majority of the fragment distribution is between 100 to 300 base pairs.  RNA-seq samples are usually fragmented after poly-A selection and before first strand synthesis.<br>
* Fragmentation - All samples must be fragmented to the point where the majority of the fragment distribution is between 100 to 300 base pairs.  RNA-seq samples are usually fragmented after poly-A selection and before first strand synthesis.<br>
-
* End Repair and 3’ dA Addition – Any damage end damage that may have occurred during fragmentation is repaired and then an extra A is added to all 3’ ends to increase efficiency during ligation. <br>
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* End Repair and 3’ dA Addition – Any end damage that may have occurred during fragmentation is repaired and then an extra A is added to all 3’ ends to increase efficiency during ligation. <br>
* Adapter ligation – Partial Illumina adapter sequences are ligated to the sample fragments, a total addition of 66bp between the two ends. Each adapter sequence has a T-overhang to increase efficiency. It is very important to maintain a good ratio of sample material to adapter. Illumina recommends a 1:10 ratio, to avoid the presence of primer-dimer after enrichment.<br>
* Adapter ligation – Partial Illumina adapter sequences are ligated to the sample fragments, a total addition of 66bp between the two ends. Each adapter sequence has a T-overhang to increase efficiency. It is very important to maintain a good ratio of sample material to adapter. Illumina recommends a 1:10 ratio, to avoid the presence of primer-dimer after enrichment.<br>
-
* Size Selection – This step, is designed so that only the fragments within the desired size range (generally ~200–400bp) are included in the final library.  Size selection is very important: fragments that are too large will create oversized, overlapping clusters on the Illumina flowcell and can result in a loss of read count in the final data. Size selection is usually performed using an agarose gel.<br>
+
* Size Selection – This step is designed so that only the fragments within the desired size range (generally ~200–400bp) are included in the final library.  Size selection is very important: fragments that are too large will create oversized, overlapping clusters on the Illumina flowcell and can result in a loss of read count in the final data. Fragments much smaller than the optimal size range may include residual primers and primer-dimers. Size selection is usually performed using an agarose gel.<br>
-
* Enrichment – The DNA is PCR-amplified using the complete primer construct required for binding and clustering on the flowcell. This adds 53bp of adapter sequence total between the two ends, for a final adapter length of 119bp.  It is very important to optimize the number of PCR cycles so that there is sufficient material for clustering but limited PCR bias.<br>
+
* Enrichment – The DNA is PCR-amplified using the complete primer constructs required for binding and clustering on the flowcell. This adds 53bp of adapter sequence total between the two ends, for a final adapter length of 119bp.  It is very important to optimize the number of PCR cycles so that there is sufficient material for clustering while limiting PCR biases.<br>
Traditional Illumina protocols can be found on our [[BioMicroCenter:Protocols| protocols page]]. <br><br><br>
Traditional Illumina protocols can be found on our [[BioMicroCenter:Protocols| protocols page]]. <br><br><br>
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== Barcoding ==
+
 
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Our '''[[BioMicroCenter:DNA Sample Preparation|BMC Fragmented DNA sample prep (SPRI and enrichment)]]''' service includes [[BioMicroCenter:Multiplex|molecular barcoding]] to allow sequencing of multiple samples per lane. Nextera DNA prep and RNAseq-prep may include barcoding at the user's option.
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Revision as of 09:27, 2 July 2012

Image:BioMicroCenter-header6.jpg

Sample Preparation available in BioMicro
Sample Preparation available in BioMicro

Generating high-quality data on the Illumina sequencing platform requires high-quality libraries. The BMC currently offers library preparation services for a variety of starting materials. Prior to sequencing, all samples must pass the BioMicro Center’s Sequencing Quality Control process, which verifies selection of inserts of a desired size and correct ligation of Illumina adapters.
Sample Prep questions can be directed to Shmulik Motola.

Contents

Sample Prep Services

DNA

There are two options when submitting DNA for library prep depending on the type of sample submitted.

Fragmented DNA - Samples will be pre-QC'd by BioAnalyzer, run on the SPRI-works system using BioMicro Center adapters (with size-selection at the user's option), enriched using BioMicro Center PCR primers (including molecular barcodes for multiplexing), final-QC'd, and either submitted for Illumina sequencing or returned to the user for their own sequencing. Users may also chose an a la carte service where only SPRI is preformed.

BMC Intact DNA sample prep (NEXTERA) - Samples will be pre-QC'd by Nanodrop, processed using the Epicentre Nextera kit, enriched using Nextera PCR primers (including molecular barcodes if requested), final-QC'd on the BioAnalyzer and either submitted for Illumina sequencing or returned to the user for their own sequencing. At least 50ng of DNA is required for Nextera. Samples for Nextera cannot be pre-QC'ed on the BioAnalyzer because the large fragments will clog the microchannels.

Size Selection - For samples requiring tight size selection, amplified libraries can be size selected using the PippinPrep prior to sequencing.

RNA

There are several options when submitting RNA for library prep. These options are largely dictated by the source of the RNA, the quality of the RNA and the amount of RNA available.In addition, strand specific information is available for some methods and tight size selection is available for all methods.

mRNA-seq - Samples will be pre-QC'd on the BioAnalyzer, poly-A purified and converted to cDNA using the Illumina Tru-Seq protocol, run on the SPRI-works system using BioMicro Center adapters, enriched using BioMicro Center PCR primers, and submitted for Illumina sequencing.

Strand Specific mRNA-seq - Samples will be pre-QC'd on the BioAnalyzer, poly-A purified and converted to cDNA using the dUTP 2nd strand marking protocol outlined in J. Levin et al 2010, run on the SPRI-works system using BioMicro Center adapters, enriched using BioMicro Center PCR primers, and submitted for Illumina sequencing.

Low Input mRNA-seq - Samples will be pre-QC'd on the BioAnalyzer, poly-A purified and converted to cDNA using the Ultra Low Input mRNAseq Kit from Clontech, run on the SPRI-works system using BioMicro Center adapters, enriched using BioMicro Center PCR primers, and submitted for Illumina sequencing.

|Chromatin

ChIP-Seq: Sonicated chromatin will undergo IP on the IPstar. RNase A, Proteinase K, Phenol:Chloroform extraction and purification will be performed off of the instrument and submitted for QC on the BioAnalyzer. These ChIP samples may be picked up for qPCR or sent directly to SPRI for library construction and sequencing (options are listed on submission forms). Users will be contacted if ChIP samples fail QC. These samples will not be submitted to SPRI-works but will be charged for the IP.

Barcoding

Our BMC Fragmented DNA sample prep (SPRI and enrichment) service includes molecular barcoding to allow sequencing of multiple samples per lane. Nextera DNA prep and RNAseq-prep may include barcoding at the user's option.

Submission

Unless otherwise instructed, the BMC reserves 50% of submitted DNA in case of failures during sample prep. If you would like us to utilize the full sample, please indicate it in the notes section.

DNA– DNA samples must be submitted in either water or TE. Although the SPRIworks robot is not highly sensitive to the amount of DNA input, care must be taken to submit an appropriate amount of adapter to ensure efficient ligation. If you select the Nextera kit instead of the SPRIworks, we will require 50ng of DNA input.

RNA – Total RNA samples must be submitted in water or TE. The quantity of total RNA needs to be between 0.1-4ug for Illumina TruSeq, 1ug-4ug for strand specific RNAseq, >100pg for Low Input RNAseq.

Chromatin – Sonicated chromatin must be submitted at 5-7 million cells per 200uL volume. Antibodies must be submitted at the same time(at least 3ug of antibody per IP). Chromatin should be prepared on the SAME DAY as the ChIP whenever possible. Full details about isolation methods can be found on the chromatin IP page.

Sample Submission Forms


QC

Good quality control is crucial for optimizing the number of reads and quality of data produced by the sequencers. For more information on QC methods and protocols please visit the Sequencing Quality Control page.


Workflow

  • Fragmentation - All samples must be fragmented to the point where the majority of the fragment distribution is between 100 to 300 base pairs. RNA-seq samples are usually fragmented after poly-A selection and before first strand synthesis.
  • End Repair and 3’ dA Addition – Any end damage that may have occurred during fragmentation is repaired and then an extra A is added to all 3’ ends to increase efficiency during ligation.
  • Adapter ligation – Partial Illumina adapter sequences are ligated to the sample fragments, a total addition of 66bp between the two ends. Each adapter sequence has a T-overhang to increase efficiency. It is very important to maintain a good ratio of sample material to adapter. Illumina recommends a 1:10 ratio, to avoid the presence of primer-dimer after enrichment.
  • Size Selection – This step is designed so that only the fragments within the desired size range (generally ~200–400bp) are included in the final library. Size selection is very important: fragments that are too large will create oversized, overlapping clusters on the Illumina flowcell and can result in a loss of read count in the final data. Fragments much smaller than the optimal size range may include residual primers and primer-dimers. Size selection is usually performed using an agarose gel.
  • Enrichment – The DNA is PCR-amplified using the complete primer constructs required for binding and clustering on the flowcell. This adds 53bp of adapter sequence total between the two ends, for a final adapter length of 119bp. It is very important to optimize the number of PCR cycles so that there is sufficient material for clustering while limiting PCR biases.

Traditional Illumina protocols can be found on our protocols page.







Pricing and Priority

Full pricing information is available on our price list.

Priority for Illumina sample prep is currently given to labs associated with the BioMicro Center Core departments. We are able offer our services to other MIT and non-MIT users as space allows.


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