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| [[Image:Presentationbest.jpg|right|400px]] | | [[Image:BMC_samplePrep_pipes.png|thumb|right|400px|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 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>Sample Prep questions can be directed to [[BioMicroCenter:People|Michael Gravina and Ryan Sinapius]]. | | 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 Services==
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| === DNA ===
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| There are three options when submitting DNA for library prep:
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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.
| | == Short Read Library Prep Services== |
| | Please follow the links in the table below for more information about our library preparation offerings. |
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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.
| | {| style="wikitable" border=1 |
| | ! DNA |
| | ! RNA |
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| | | '''[[BioMicroCenter:DNA_LIB|Standard DNA Methods include]]''' |
| | * LMPCR methods |
| | ** NEB UltraII |
| | * Tagmentation Methods |
| | ** Nextera XT |
| | ** Nextera Flex |
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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:RNA_LIB|Standard RNA Methods include]]''' |
| | * NEB UltraII <br> polyA stranded method |
| | * NEB ribosomal depletion (NEB or Lexogen for Bacteria) <br> stranded rRNA depletion |
| | * SMARTer_Low-Input <br> Low input polyA based |
| | * Clontech ZapR <br> Low input rRNA depletion based. |
| | * smallRNA prep |
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| | | '''[[BioMicroCenter:DNA_HTL|High Throughput DNA Methods include]]''' |
| | * Amplicon / 16S |
| | * NexteraXT |
| | * low input NexteraXT |
| | * Nextera Flex |
| | * NEB UltraII |
| | | '''[[BioMicroCenter:RNA_HTL|High Throughput RNA Methods include]]''' |
| | * PolyA (NEB UltraII) |
| | * NEB ribosomal depletion |
| | * High Throughput 3' Digital Gene Expression (HT3DGE) |
| | * SMARTseq v2 |
| | * ZapR |
| | |} |
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| === RNA === | | == OTHER SERVICES == |
| | | === SIZE SELECTION === |
| '''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.
| | The BMC offers [[BioMicroCenter:PippinPrep|PippinPrep Size Selection]] post library construction if custom insert sizes are needed. |
| | | === QUALITY CONTROL === |
| === Chromatin === | | All NGS libraries are quality controlled prior to use. The BioMicro Center offers two 'flavors' of quality control for Illumina libraries.<BR><BR> |
| | | Standard quality control includes analysis on the [[BioMicroCenter:QC#AATI_FRAGMENT_ANALYZER|Fragment Analyzer]] and quantification by four point [[BioMicroCenter:RTPCR|qPCR]] quantification for each sample. This confirms both the sizing as well as the presence of Illumina adapters on the libraries.<BR><BR> |
| 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.
| | Rapid Quality Control is less expensive and quicker. It includes analysis on the [[BioMicroCenter:QC#AATI_FRAGMENT_ANALYZER|Fragment Analyzer]] and quantification by PicoGreen on the [[BioMicroCenter:Varioskan|Varioskan]] for each sample. Final pools are quantified by [[BioMicroCenter:RTPCR|qPCR]] before loading. While this method is significantly faster and less expensive, we cannot guarantee pool balance with it. |
| | | <BR><BR>[[BioMicroCenter:PREP_TEST]] |
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| '''[[BioMicroCenter:Forms#Downloads|Sample Submission Forms]]'''<br><br><br>
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| ==Submission== | |
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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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| '''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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| '''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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| 1. Resuspend fixed cells in 10mL of [[LB1|LB1]] <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 ==
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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>
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| ==Workflow==
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| [[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>
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| * 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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| * 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>
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| * 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>
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| * 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>
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| 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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| == Pricing and Priority ==
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| Full pricing information is available on [[BioMicroCenter:Pricing|our price list]].<br>
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| Priority for Illumina sample prep is currently given to labs associated with the BioMicro Center [[BioMicroCenter:CoreDeps|Core departments]]. We are able offer our services to other MIT and [[BioMicroCenter:FAQ#NON_MIT_USERS|non-MIT]] users as space allows. <br><br><br>
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