Editing BioMicroCenter:Illumina Sequencing (section)

== Illumina Massively Parallel Sequencing ==
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  !Service 
  !Illumina Sequencing
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  |INPUT || Illumina libraries
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  |MIN VOLUME || 12 uL NextSeq/MiSeq per flow cell <br> 12 uL NovaSeq per lane
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  |MIN CONCENTRATION || 2nM* (~0.4ng/uL for a 300bp library)<BR> Can handle samples to 0.2nM, but read count not guaranteed
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  |INCLUDED SERVICES 
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* Quality Control:Fragment Analyzer and qPCR
* Illumina Sequencing 
* Demultiplexing
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  |ADDITIONAL SERVICES ||
* Quality Control
* Illumina library preparation
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  |DATA FORMATS 
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*FASTQ 
*BCL (stored 30d)
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  |QUALITY CONTROL 
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* [https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ FASTQC]
* Basic run metrics (alignment rate, complexity)
* Basic RNAseq metrics (where applicable)
* Basic paired end metrics (where applicable)
* Contamination checks
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  |PRICING || [[BioMicroCenter:Pricing#HISEQ2000_SEQUENCING|LINK]]
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  |SUBMISSION 
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* MIT - [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=3863 ilabs] 
* External Users - [[BioMicroCenter:Forms|form]]
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Illumina sequencing-by-synthesis is the primary workhorse of the BioMicro Center. These instruments produce millions of reads simultaneously and are used in a very broad spectrum of applications. The Center currently supports 1 NovaSeq 6000, 1 NextSeq500, and 1 MiSeq. In addition, we have access to additional sequencing instrumentation through collaborations with the Whitehead Genome Technologies Core and other local academic core facilities. <BR><BR>

===Typical Workflow===
Illumina sequencing at the core begins with library quality control, during which the Center verifies the anchor elements and insert size using [[BioMicroCenter:RTPCR|qPCR]] and the [[BioMicroCenter:QC#AATI_FRAGMENT_ANALYZER|Fragment Analyzer]]. Users may elect to bypass this step if they provide the sample concentration and the concentration they would like to load at. Samples are then entered into the sequencing queue. Typical queues in the BioMicro Center are short, rarely exceeding 1-2 weeks, and samples are frequently run within a couple days. <B>We do guarantee a minimum number of reads per lane provided if: a) BMC performed the QC, b) the samples are high-complexity, especially in the first few nucleotides, and c) the samples are at least 2nM.</B> <br>

Illumina sequencing through the BioMicro Center is only available in full lanes and not on a per read basis. You are welcome to collaborate with other laboratories in order to share a lane. However, please be sure to minimize any possibility of cross-contamination of indexes as well as [https://www.illumina.com/science/education/minimizing-index-hopping.html index crosstalk.] 

When ordering lanes, please be aware that only full flowcells will be sequenced. More lanes may be purchased to facilitate sequencing. The wait time for individual lanes can vary widely. Full flowcells have priority for sequencing.<BR><BR>

===Data Handling===
Following sequencing, data is handled using a custom analytical pipeline. If an index was provided, samples are split by index and identified by DNA-ID. [https://en.wikipedia.org/wiki/FASTQ_format FASTQ] files and other desired formats will be placed in a delivery folder based on the project name, along with several quality control checks done for the sequencing data. We will provide an initial review of your project where we do try to identify any issues including incorrect indexes, sample contaminants, etc. Please note that our pipelines are built to find problems and are NOT designed to provide an initial analysis of your data. These analyses are built for speed, simplicity, and are not tuned in any way for your samples. We are always happy to discuss these with you. Data will be available in your lab folder for 90 days post delivery, after which most of the data is deleted. FASTQ file will still be downloadable for two years but is delivered on a case by case basis.<BR><BR>
===Custom Sequencing===
Users may elect to prep their samples with custom oligos. If this is the case, custom sequencing oligos must be provided along with the samples at the time of submission. At least 15uL of each kind custom sequencing primer at 100 uM should be submitted per lane of MiSeq, NextSeq or NovaSeq. At least 30uL of each kind of custom sequencing primer at 100uM should be provided per NovaSeq flowcell. It is strongly recommended to contact biomicro@mit.edu before planning a custom library preparation. <BR><BR>


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<BR><BR>
Additional services available:
* [[BioMicroCenter:Illumina_Library_Preparation|Illumina library preparation]]
* [[BioMicroCenter:BioInformaticsStaff|Bioinformatics Support]]
* [[BioMicroCenter:Servers|Data storage and Computation]]
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