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== INTACT GENOMES == === Nextera Flex === {| |- style="vertical-align: top;" | style="width:500px;" | {| class="wikitable" border=1 |- ! style="text-align: center; width: 150px;" | Parameter ! style="text-align: center; width: 300px" | General requirements ! style="text-align: center; width: 300px" | [[BioMicroCenter:DNA_HTL#DNA_Pre-load_Submissions|Pre-load requirements]] |- | style="text-align: center; height: 2em;" | SAMPLE INPUT | colspan="2" style="text-align: center; height: 2em;" | Intact DNA and large amplicons |- | style="text-align: center; height: 2em;" | RANGE OF INPUT | style="text-align: center; height: 2em;" | 1ng - 50ng | style="text-align: center; height: 2em;" | Samples all [[BioMicroCenter:DNA_HTL#Normalization|normalized]] to same concentration in 3µL (330pg/µL - 16.5ng/µL, *higher is better) |- | style="text-align: center; height: 2em;" | SUBMISSION VOLUME | style="text-align: center; height: 2em;" | >10µL 10 mM Tris Buffer | style="text-align: center; height: 2em;" | Exactly 3µL 10 mM Tris Buffer |- | style="text-align: center; height: 2em;" | UNIT | colspan="2" style="text-align: center; height: 2em;" | 24 samples <BR> 96 samples |- | style="text-align: center; height: 2em;" | PLATE SETUP | style="text-align: center; height: 2em;" | Samples should be arrayed by column in a 96-well full-skirt plate (Axygen) | style="text-align: center; height: 2em;" | Samples arrayed by column in [[BioMicroCenter:DNA_HTL|1st quadrant]] of 384-well hard-shell plate (BioRad) |- | style="text-align: center; height: 2em;" | SEQUENCING RECOMMENDATIONS | colspan="2" style="text-align: center; height: 2em;" | All platforms except 40SE HiSeq |- | style="text-align: center; height: 2em;" | INDEX AVAILABILITY | colspan="2" style="text-align: center; height: 2em;" | 384 [[BioMicroCenter:DNA_HTL#Unique_versus_Combinatorial_Dual_Indexing|Unique Dual Indexes]] <br> [[BioMicroCenter:DNA_HTL#Unique_versus_Combinatorial_Dual_Indexing|Combinatorial Dual Indexes]] ''not available'' |- | style="text-align: center; height: 2em;" | INCLUDED | colspan="2" style="text-align: center; height: 2em;" | Library preparation <BR> Spot check of final libraries |- | style="text-align: center; height: 2em;" | ADDITIONAL SERVICES AVAILABLE | style="text-align: center; height: 2em;" | Sample QC <BR> Sample cleaning <BR> Sample arraying <BR> Sample re-prep | style="text-align: center; height: 2em;" | [[BioMicroCenter:DNA_HTL#DNA_Pre-load_Submissions|N/A]] |- | style="text-align: center; height: 2em;" | SUBMISSION FORM | colspan="2" style="text-align: center; height: 2em;" | MIT - [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=3863 ilabs] <BR> External - [[BioMicroCenter:Forms|form]] |- | style="text-align: center; height: 2em;" | PRICING | colspan="2" style="text-align: center; height: 2em;" | [[BioMicroCenter:Pricing#ILLUMINA_LIBRARY_-_DNA|LINK]] |} | <br> :Unlike XT, Nextera Flex, also known as Illumina DNA Prep, utilizes bead-linked transposomes (BLT) to tagment and generate libraries from intact DNA samples. These BLTs control sizing during the tagmentation reaction by sterics, producing a much more consistent libraries with larger size distribution which makes it ideal for larger paired-end runs on sequencers. :Nextera Flex is currently run at a 1/10th miniaturization scale on the Mosquito HV with a broader dynamic range of input (1ng - 50ng) compared to XT. Nextera Flex self-normalizes at a certain input (if sample concentration is 3ng/µL or greater) and submissions do not have to be normalized so long as that threshold is met. Up to 384 libraries can be multiplexed using Nextera unique dual-indexes (UDI) which helps to avoid issues with 'barcode hopping'. ::::[[IMAGE:Flex_Diagram.jpg | 500px]] ::::::[[IMAGE:Flex_Trace.jpg | 300px]] | |} === Nextera XT === {| |- style="vertical-align: top;" | style="width:500px;" | {| class="wikitable" border=1 |- ! style="text-align: center; width: 150px;" | Parameter ! style="text-align: center; width: 300px" | General requirements ! style="text-align: center; width: 300px" | [[BioMicroCenter:DNA_HTL#DNA_Pre-load_Submissions|Pre-load requirements]] |- | style="text-align: center; height: 2em;" | SAMPLE INPUT | colspan="2" style="text-align: center; height: 2em;" | Intact DNA and large amplicons |- | style="text-align: center; height: 2em;" | RANGE OF INPUT | style="text-align: center; height: 2em;" | > 0.2ng/µL | style="text-align: center; height: 2em;" | Samples [[BioMicroCenter:DNA_HTL#Normalization|normalized]] to 0.2ng/µL |- | style="text-align: center; height: 2em;" | SUBMISSION VOLUME | style="text-align: center; height: 2em;" | >10µL 10 mM Tris Buffer | style="text-align: center; height: 2em;" | Exactly 5µL 10 mM Tris Buffer |- | style="text-align: center; height: 2em;" | UNIT | colspan="2" style="text-align: center; height: 2em;" | 24 samples <BR> 96 samples |- | style="text-align: center; height: 2em;" | PLATE SETUP | style="text-align: center; height: 2em;" | Samples should be arrayed by column in a 96-well full-skirt plate (Axygen) | style="text-align: center; height: 2em;" | Samples arrayed by column in [[BioMicroCenter:DNA_HTL#Quadrant_Layout|1st quadrant]] of a 384-well LVSD plate (Available at BMC) |- | style="text-align: center; height: 2em;" | SEQUENCING RECOMMENDATIONS | colspan="2" style="text-align: center; height: 2em;" | 40SE HiSeq |- | style="text-align: center; height: 2em;" | INDEX AVAILABILITY | colspan="2" style="text-align: center; height: 2em;" | 16 [[BioMicroCenter:DNA_HTL#Unique_versus_Combinatorial_Dual_Indexing|Unique Dual Indexes]] <br> 384 [[BioMicroCenter:DNA_HTL#Unique_versus_Combinatorial_Dual_Indexing|Combinatorial Dual Indexes]] |- | style="text-align: center; height: 2em;" | INCLUDED | colspan="2" style="text-align: center; height: 2em;" | Library preparation <BR> Spot check of final libraries |- | style="text-align: center; height: 2em;" | ADDITIONAL SERVICES AVAILABLE | style="text-align: center; height: 2em;" | Sample QC <BR> Sample cleaning <BR> Sample arraying <BR> Sample re-prep | style="text-align: center; height: 2em;" | [[BioMicroCenter:DNA_HTL#DNA_Pre-load_Submissions|N/A]] |- | style="text-align: center; height: 2em;" | SUBMISSION FORM | colspan="2" style="text-align: center; height: 2em;" | MIT - [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=3863 ilabs] <BR> External - [[BioMicroCenter:Forms|form]] |- | style="text-align: center; height: 2em;" | PRICING | colspan="2" style="text-align: center; height: 2em;" | [[BioMicroCenter:Pricing#ILLUMINA_LIBRARY_-_DNA|LINK]] |} | <br> :Nextera XT is a solution-based "tagmentation" chemistry used for the preparation of intact DNA samples to generate libraries with smaller insert sizes which is best suited for 40bp single-end runs on sequencers. Nextera XT has been miniaturized to a 1/12th reaction volume on our Mosquito HV from minimal low sample concentration (0.2ng/µL or greater) and up to 384 libraries can be run on a single sequencing lane using dual indexes. :The Mosquito HV allows for libraries to be prepared at decreased volumes which results in significantly reduced costs but also lowers library complexity. As such, it is most suitable for single cell and amplicon analysis. De novo work should not be done using Nextera XT preps on the Mosquito HV. ::[[IMAGE:Nextera_Illumina_fig1.jpg | 200px]][[IMAGE:XT_Trace.jpg | 250px]] | |}
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