Editing BioMicroCenter:Illumina Sequencing

{{BioMicroCenter}}

The MIT BioMicro Center has six high-throughput Illumina sequencers including one NovaSeq 6000, one MiSeq, and one NextSeq 500. We support a wide variety of applications, such as ChIP-Seq, miRNA sequencing and RNA-seq. Each lane has the potential to accommodate dozens of barcoded samples (depending on sequence complexity and desired coverage). [[BioMicroCenter:CoverageCalculations|Read lengths]] vary, depending on users, between 20nt and 325nt per end. <br><br>

== Illumina Massively Parallel Sequencing ==
{|
 |- style="vertical-align: top;"
 |style="width: 400px;"|
 {| class="wikitable" border=1
  !Service 
  !Illumina Sequencing
  |-
  |INPUT || Illumina libraries
  |-
  |MIN VOLUME || 12 uL NextSeq/MiSeq per flow cell <br> 12 uL NovaSeq per lane
  |-
  |MIN CONCENTRATION || 2nM* (~0.4ng/uL for a 300bp library)<BR> Can handle samples to 0.2nM, but read count not guaranteed
  |-
  |INCLUDED SERVICES 
  | 
* Quality Control:Fragment Analyzer and qPCR
* Illumina Sequencing 
* Demultiplexing
  |-
  |ADDITIONAL SERVICES ||
* Quality Control
* Illumina library preparation
  |-
  |DATA FORMATS 
  |
*FASTQ 
*BCL (stored 30d)
  |-
  |QUALITY CONTROL 
  |
* [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
  |-
  |PRICING || [[BioMicroCenter:Pricing#HISEQ2000_SEQUENCING|LINK]]
  |-
  |SUBMISSION 
  |
* MIT - [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=3863 ilabs] 
* External Users - [[BioMicroCenter:Forms|form]]
  |-
 |}
 |
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>


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

== Illumina Platforms ==
{| class="wikitable" border=1 
 !width=100| SPEC
 !width=250| MiSeq
 !width=250| NextSeq500
 !width=250| NovaSeq6000
 !width=250| NovaSeqXplus
 |-
 |'''SEQUENCER'''
 |[[Image:BMC_miseq.jpg|center|200px]]
 |[[Image:BMC_Next.jpg|center|200px]]
 |[[Image:BMC_Nova6000.jpg|center|200px]]
 |[[Image:BMC_NovaXplus.jpg|center|150px]] 
 |-
 | '''READS/LANE'''<BR> Low number is minimum per lane for standard Illumina libraries.
 | 
* v2: 8-15M 
* v3: 12-25M
* Nano (v2 Reagents only): 1M
 | 
* High output: 250-500M
 | 
* SP, S1, S2 (2 lanes): 650M, 1.3B, 3.3B 
*S4 (4 lanes): 8B
 |
*10B (8 lanes)
*25B (8 lanes or full flowcell)
 |-
 |'''LENGTHS AVAILABLE'''
 |
*'''v2:''' 75nt, 300nt, 500nt
*'''v3:''' 150nt, 600nt
 |
*75nt, 150nt, 300nt
 |
*100nt, 200nt, 300nt, 500nt(SP only)
 |
*100nt(25B full FC only),300nt
 |-
 |'''USES'''
 |
* Small genome resequencing
* Targeted resequencing
* 16S Metagenomes
* smRNA
* DeNovo Sequencing
* Low coverage
 |
* SNP detection
* Exome sequencing
* Splicing analysis in RNAseq
* Medium coverage
* DeNovo Sequencing
* Metagenomics
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* scRNA sequencing for many cells
* Exome sequencing
* High coverage
 |
* Highest coverage
 |-
 |'''KEY NOTES'''
 |
*Best for low complexity libraries
*Suitable for barcode counting
 |
*2 color chemistry - G=dark just like NovaSeq
*Struggles with low complexity libraries
*Struggles with large libraries
 |
*Patterned flowcell
*Struggles with low complexity libraries
*Lane by Lane
 |
*Patterned flowcell
*Struggles with low complexity libraries
*Lane by Lane
 |-
 |'''DONATED BY'''
 |Drs. Chris Love, Michael Birnbaum and the Dept. of Biological Engineering.
 |Drs. Penny Chisholm, Doug Lauffenburger, Myriam Heiman, Li-Huei Tsai and the Dept. of Biology and the Koch Institute.
 |Drs. Manolis Kellis,  Li-Huei Tsai and MIT, The MIT Stem Cell Initiative and the Dept. of Biology and the Koch Institute.
 |Collaborations with local academic cores. 
|}


<!-- commenting GA out 2/2/17 NK === Genome Analyzer IIx ===
HiSeq2000s donated by Drs. Penny Chisholm and Chris Burge and HHMI

[[Image:GAIIxcollage.jpg|right|200px]] The Genome Analyzer II (GAII) is the oldest sequencers in the BioMicro Center and remain the most flexible. The newer generations of Illumina sequencers have been designed with increasing focus on clinical applications and have removed some of the "hands on" aspects of the older GAIIs. The GAIIs remain the only sequencers where the actual images of the flowcell can be reprocessed for example. The GAII/IIx can produce 20-40m reads per lane passing filter and typically runs read lengths of 36-150nt per side.<BR><BR>
With the addition of the MiSeq, we have reworked how we are processing GAII flowcells. We have been able to create [[BioMicroCenter:PartialFlowcells|'''partial flowcells''']] on the GAII by altering recipes. This has allowed us to move from a model like the HiSeq where we need a full flowcell before we run to a model where we can run as soon as the samples pass quality control, more like the MiSeq. However, unlike the MiSeq, we can run multiple lanes at once. Some critical caveats: First, these methods are not supported by Illumina so we cannot offer to replace failed runs. Second, unlike the HiSeq, the PhiX lane is *not* included. You must choose to sequence a lane of PhiX if you want to do control normalization. Finally, this service is completely "a la carte" so the pricing schema is quite different. <BR><BR>
{| border=1 align="right"
 ! # of Lanes
 !width=75| cycles per day
 !width=75| cycles per kit
 |-align="center"
 | 8 
 | 24 
 | 42
 |-align="center"
 | 4 
 | 36 
 | 66/33*
 |-align="center"
 | 2 
 | 48 
 | 106/54*
 |-align="center"
 | 1 
 | 72 
 | 140/81*
|-
|colspan="3" align=center |&#42;''Second number pertains to reads greater than 40 nt.''
|}

Using fewer lanes on each flowcell has allowed us to decrease the cycle time by not imaging all the lanes. In a typical 8 lane run, 20 minutes is spent doing chemistry followed by 40 minutes of imaging (each lane takes ~5 minutes to image). Therefore, a 2 lane flowcell runs twice as fast as an 8 lane flowcell. Also, since the chemistry is not running in to all of the lanes, each sequencing kit can go to a longer read length. The relationships are summarized in the chart on the left. Pricing is set on the number of lanes you are using, the number of days you are running the GAII, and the number of sequencing kits you are using. For example, if you wanted to run a 75+75 PE flowcell using 2 lanes, the cost would be the initial cost for the 2 lane PE flowcell plus an additional 3 days (one day is included in the original price) plus two additional sequencing kits. The last kit would not be completely used up (you would have an extra 18nt left that would be thrown away).<BR><BR> 

The GAII/GAIIx is ideal for:
* Unusual read lengths
* Protocol Prototyping
* Non-standard assays such as HITS-FLIP

''The Genome Analyzer IIs were donated to the BioMicro Center by Drs. Penny Chisholm, Chris Burge, Ernest Fraenkel and the Dept of Biology with contributions from many others ''
 !width=200| GAII/IIx:  Boris, Natasha, etc 20-40m reads 1 to 8 lanes 24-72 nt/day max read length 80+80 in lane by lane
THE GA IS DEAD LONG LIVE THE GA Jack Daniels is DEAD LONG LIVE JACK DANIELS-->