Editing BioMicroCenter:Illumina Sequencing (section)

== 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
 |
* 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-->