BioMicroCenter:Illumina Sequencing: Difference between revisions
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The MIT BioMicro Center | The MIT BioMicro Center operates a MiSeq i100 and supports NovaSeqX Plus sequencing from Illumina. We support a wide variety of applications, such as ChIP-Seq, miRNA sequencing and RNA-seq. | ||
== Illumina Massively Parallel Sequencing == | == Illumina Massively Parallel Sequencing == | ||
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|INPUT || Illumina libraries | |INPUT || Illumina libraries | ||
|- | |- | ||
|MIN VOLUME || 40uL | |MIN VOLUME || 40uL MiSeq i100 <BR> 110 uL NovaSeqXplus per lane | ||
|- | |- | ||
|MIN CONCENTRATION || 2nM* (~0.4ng/uL for a 300bp library) | |MIN CONCENTRATION || 2nM* (~0.4ng/uL for a 300bp library) | ||
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Illumina sequencing-by-synthesis is the primary workhorse of the BioMicro Center. These instruments produce millions to billions of reads simultaneously and are used in a very broad spectrum of applications. The Center currently | Illumina sequencing-by-synthesis is the primary workhorse of the BioMicro Center. These instruments produce millions to billions of reads simultaneously and are used in a very broad spectrum of applications. The Center currently operates a MiSeq i100 walkup sequencer and supports NovaSeqX Plus through collaboration with other local academic core facilities. <BR><BR> | ||
===Typical Workflow=== | ===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 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 pool samples. 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.] | 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 pool samples. 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 ASAP. 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> | When ordering lanes, please be aware that only full flowcells will be sequenced ASAP. 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=== | ===Data Handling=== | ||
Following sequencing, data is handled using a custom analytical pipeline. If libraries were made in the Center or 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 to your samples. We are always happy to discuss these results 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> | Following sequencing, data is handled using a custom analytical pipeline. If libraries were made in the Center or 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 to your samples. We are always happy to discuss these results 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> | ||
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* [[BioMicroCenter:Servers|Data storage and Computation]] | * [[BioMicroCenter:Servers|Data storage and Computation]] | ||
|} | |} | ||
== Illumina Platforms == | == Illumina Platforms == | ||
{| class="wikitable" border=1 | {| class="wikitable" border=1 | ||
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* 5M | * 5M | ||
* 25M | * 25M | ||
* 50M | |||
* 100M | |||
| | | | ||
*10B (8 lanes) | *10B (8 lanes) | ||
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|'''LENGTHS AVAILABLE''' | |'''LENGTHS AVAILABLE''' | ||
| | | | ||
* 100nt, 300nt, 600nt | * 100nt, 300nt, 600nt, 1000nt | ||
| | | | ||
*100nt(25B full FC only),300nt | *100nt(25B full FC only),300nt | ||
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|Collaborations with local academic cores. | |Collaborations with local academic cores. | ||
|} | |} | ||
<!-- commenting GA out 2/2/17 NK === Genome Analyzer IIx === | <!-- commenting GA out 2/2/17 NK === Genome Analyzer IIx === | ||
HiSeq2000s donated by Drs. Penny Chisholm and Chris Burge and HHMI | 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> | [[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> | 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> | ||
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|colspan="3" align=center |*''Second number pertains to reads greater than 40 nt.'' | |colspan="3" align=center |*''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> | 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: | The GAII/GAIIx is ideal for: | ||
* Unusual read lengths | * Unusual read lengths | ||
* Protocol Prototyping | * Protocol Prototyping | ||
* Non-standard assays such as HITS-FLIP | * 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 '' | ''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 | !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 and Samuel Adams are DEAD LONG LIVE the HiSeq2000s Lucille and George are dead LONG LIVE NEXTSEQ 500s MiAmore is dead LONG LIVE THE MISEQ RUO PacBio is dead LONG LIVE THE SEQUEL II PromethION 24 is dead LONG LIVE THE P24 --> | THE GA IS DEAD LONG LIVE THE GA Jack Daniels and Samuel Adams are DEAD LONG LIVE the HiSeq2000s Lucille and George are dead LONG LIVE NEXTSEQ 500s MiAmore is dead LONG LIVE THE MISEQ RUO PacBio is dead LONG LIVE THE SEQUEL II PromethION 24 is dead LONG LIVE THE P24 --> | ||
Revision as of 03:34, 18 March 2026
HOME -- SEQUENCING -- LIBRARY PREP -- HIGH-THROUGHPUT -- COMPUTING -- OTHER TECHNOLOGY
The MIT BioMicro Center operates a MiSeq i100 and supports NovaSeqX Plus sequencing from Illumina. We support a wide variety of applications, such as ChIP-Seq, miRNA sequencing and RNA-seq.
Illumina Massively Parallel Sequencing
|
Illumina sequencing-by-synthesis is the primary workhorse of the BioMicro Center. These instruments produce millions to billions of reads simultaneously and are used in a very broad spectrum of applications. The Center currently operates a MiSeq i100 walkup sequencer and supports NovaSeqX Plus through collaboration with other local academic core facilities. Typical WorkflowIllumina sequencing at the core begins with library quality control, during which the Center verifies the anchor elements and insert size using qPCR and the 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. 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. 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 pool samples. However, please be sure to minimize any possibility of cross-contamination of indexes as well as index crosstalk. When ordering lanes, please be aware that only full flowcells will be sequenced ASAP. More lanes may be purchased to facilitate sequencing. The wait time for individual lanes can vary widely. Full flowcells have priority for sequencing. Data HandlingFollowing sequencing, data is handled using a custom analytical pipeline. If libraries were made in the Center or an index was provided, samples are split by index and identified by DNA-ID. 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 to your samples. We are always happy to discuss these results 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. Custom SequencingUsers 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 most Illumina sequencers. At least 30uL of each kind of custom sequencing primer at 100uM should be provided per NovaSeqXplus flowcell. It is strongly recommended to contact biomicro@mit.edu before planning a custom library preparation. |
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Illumina Platforms
| SPEC | MiSeq i100+ | NovaSeqX+ |
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| SEQUENCER |  |
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| READS/LANE Low number is minimum per lane for standard Illumina libraries. |
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| LENGTHS AVAILABLE |
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| KEY NOTES |
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| DONATED BY | MIT VPR, Drs. Gene Li, Michael Birnbaum, Dept. of Biololgy, Scott Ritterbush '89, SM '92 | Collaborations with local academic cores. |