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== Element Sequencing ==
== Element Sequencing ==
The Center currently hosts an AVITI24 platform from [https://www.elementbiosciences.com/ Element Biosciences].  The AVITI24 platform supports both sequencing and cytoprofiling. For most sequencing needs, the Center will use Element's Cloudbreak chemistry kits. Cloudbreak is a [https://www.nature.com/articles/s41587-023-01750-7#Sec1\ sequencing-by-binding method that uses the concept of avidity] to produces high quality sequencing reads.  Cloudbreak circularizes linear libraries, and then performs rolling circle amplification to generate thousands of copies of the original template on a single, linear strand.  The strand is then packaged up into what is known as a polony.  These polonies are washed with a complex of primers, polymerase, and dye-labelled avidites.  Avidites are multivalent-nucleotide-ligands on dye labeled cores capable of binding to multiple sites to form a polymerase–polymer–nucleotide complex within a polony.  After imaging, the avidites are washed away and the growing read strand incorporates a single nucleotide that is reversibly blocked on the 3' end.  The polonies are again washed with avidites, repeating the process over many cycles.  The AVITI24 can run 2 flow cells per run, each hosting 2 independently-addressable lanes.  Note, however, that the reagents that come with each kit address the entire flow cell.  In other words, while the flow cells may have 2 different libraries loaded, one in each lane, both lanes will be washed with the same set of primers.  Element currently offers kits with 150 cycles and 300 cycles, both supporting 2 lanes, as well as a 600 cycle kit that supports a single lane. With Cloudbreak's update to "Freestyle", the AVITI24 is capable of sequencing both Illumina-ready and/or Element ready libraries directly on the flow cell. High-output kits generally produce 300 to 800 million high quality read. Index hopping rates are virtually nil, limiting the need for UDI's. The AVITI24 can also perform single cell spatial imaging of RNA, proteins, phospho-proteins, and cell morphology using [https://www.elementbiosciences.com/products/aviti24/cytoprofiling/ Teton chemistry] for cytoprofiling.
The Center currently hosts an AVITI24 platform from [https://www.elementbiosciences.com/ Element Biosciences].   
 
AVITI24 platform supports both sequencing and cytoprofiling. For most sequencing needs, the Center will use Element's Freestyle chemistry kits.
=== Workflow ===
Library prep options for [https://openwetware.org/wiki/BioMicroCenter:DNA_LIB DNA] or [https://openwetware.org/wiki/BioMicroCenter:RNA_LIB RNA] are available for Element sequencing. Once Element-ready or Illumina-ready libraries have been submitted, we will verify fragment size via [https://openwetware.org/wiki/BioMicroCenter:QC AATI's Fragment Analyzer] or [https://openwetware.org/wiki/BioMicroCenter:QC Femto Pulse], and quantify the final loading pool via qPCR and Qubit.  '''We can only guarantee minimum reads per lane if: a)the BMC has performed quality control, b)the samples have high diversity, especially within the first 5 bases of read 1, and c)submitted libraries are at least 2 nM.'''
===Requirements/Things to Consider===
[[Image:AVITI FC SITES.png|left|700px]]<br clear=all>
<OL>
<LI>Polony-mapping-generation (PMG), is done on the first 5 reads of read one.  You will want to have AT LEAST these 5 positions with high diversity (roughly equal A,G,C, and T's across the flow cell per read).  However, the AVITI24 does give us the option to PMG off index 1 (sp27), with a suggested multiplex of 64 or more.  PMG can also be done upstream of the start of read 1, up to a total of 20 bases.  However, these bases will be read as dark.  You will not received data on these bases, and you will lose those cycles.  If these options can not be applied, then we will need to increase the percent phiX control spiked into the final loading sample by as much as 30% to increase the read 1 diversity.
<LI>Illumina-ready-linear libraries (have p5/p7 flow cell binding sites) are preferred as these tend sequence much more efficiently on the Freestyle flow cell, and can be applied to other platforms should the need arise.
<LI>Element-ready-linear libraries (sp5/sp27 flow cell binding sites) must have a 5'-phosphate on the sp5-end sequence in order to circularize on the AVITI.  If you choose to use Element's flow cell binding sites, sp5 and sp27, it's suggested that library prep primers are HPLC cleaned and used within a month or so as any truncated primers, as part of the primer creation process or due to degradation over time, are unlikely to circularize leading to a decrease in read count.
<LI>Library preparations involving biotin-modifications, poly-A tailing, and/or bead-based normalization will not sequence well, if at all.
<LI>Read direction will be just like MiSeq.  The index reads are sequenced first, starting with the sp27 index (which will be read off the sequencing primer site), followed by the SP5 index (which will be read off the sp5 flow cell binding sequence).  The read sequences are then read starting with read 1 (using the Read 1 primer sequence), followed by the amplification of the of the reverse strand.  Read 2 is then read using the reverse complement of the Read 2 primer sequence.
=== Data Handling ===
=== Custom Sequencing ===
{|
{|
|- style="vertical-align: top;"
|style="width: 400px;"|
  {| class="wikitable" border=1
  {| class="wikitable" border=1
   !Service  
   !width=100|Service  
   !Element Sequencing
   !width=250|Element Sequencing
   |-
   |-
   |SUBMISSION || Illumina OR Element libraries
   |SUBMISSION || Illumina OR Element libraries
Line 40: Line 26:
   |
   |
*FASTQ (stored 1 year)
*FASTQ (stored 1 year)
*BCL (stored 30d)
   |-
   |-
   |QUALITY CONTROL  
   |QUALITY CONTROL  
Line 57: Line 42:
   |-
   |-
  |}
  |}
P5/P7 anchored library (Illumina) is preferred. Element Adept libraries (SP5/SP27) require a 5'-phosphate on the forward end in order to circularize and sequence. Compared to an Illumina platform, AVITI24 reads are similar in read direction to Illumina's Forward Strand workflow for Dual Indexed Libraries.
Minimum reads per lane are guaranteed if:
*the BMC has performed quality control,
*the samples have high diversity, especially within the first 5 bases of read 1, and
*submitted libraries are at least 2 nM.
===Lane-by-lane Notes===
*The wait is variable
*Custom primers must be compatible with TruSeq, Nextera, TruSeq, smRNA and Element sequencing primers
All other requests require a full flowcell. 300PE kits only have a single lane.
* Min custom primer submission: 70 uL at 100 uM for Read 1, all other reads require 40 uL at 100 uM.


== The AVITI24 Platform ==
== The AVITI24 Platform ==
{| class="wikitable" border=1  
{| class="wikitable" border=1  
  !width=100|  
  !width=100|  
  !width=250| AVITI
  !width=250| AVITI24
  !width=350| NOTES
  !width=150| NOTES
  |-
  |-
  |'''SEQUENCER'''||[[Image:Element_AVITI.jpg|center|200px]]||
  |'''SEQUENCER'''||[[Image:Element_AVITI.jpg|center|200px]]||
  |-
  |-
  | '''READS/FLOWCELL'''<BR> Low number is minimum per lane for standard Illumina libraries.||
  | '''READS/FLOWCELL'''<BR> Low number is minimum per lane for standard P5/P7 libraries.||
* 300M HQ read pairs PF/flowcell
* 250M HQ read pairs (150 for PE300)
  |-
  |-
|'''RUN TIME'''||
|'''RUN TIME'''||
Line 75: Line 75:
  |-
  |-
|'''KITS AVAILABLE'''||
|'''KITS AVAILABLE'''||
* 2x75 Cloudbreak High
* 2x75 Cloudbreak FS High
* 2x150 Cloudbreak High
* 2x150 Cloudbreak FS High
* 2x300 Cloudbreak High
* 2x300 Cloudbreak FS High
|| - Single end supported <br> - each kit comes with 34 additional cycles
|| - Single end supported <br> - each kit comes with 34 additional cycles
|-
|'''KNOWN APPLICATIONS'''||
* Whole Genome Sequencing
* RNA Sequencing
* 16S
* Single-cell
* Target Sequencing and Enrichment
* Metagenomics
|-
|-
|'''KEY NOTES'''||
|'''KEY NOTES'''||
* 4-color chemistry
* 4-color chemistry
* Patterned flow cell
* Random flow cell
* 2-lanes per flow cell
* 2-lanes per flow cell (1 for 300PE)
* Q40+ > 80%
* Q40+ > 80%
* Low-binding surface chemistry
* Low-binding surface chemistry
* Adept Workflow for adaptation of Illumina libraries
|-
|-
|'''THANKS TO'''||
|'''THANKS TO'''||
* Element Biosciences  
* Element Biosciences / Scott Ritterbush
|}
|}
The AVITI24 can run 2 flow cells per run, each hosting 2 independently-addressable lanes.  Both lanes are washed with the same reagents.  Element currently offers kits with 150 and 300 cycles, both supporting 2 lanes, as well as a 600 cycle kit that supports a single lane. Index hopping rates are similar to non-patterned Illumina, limiting the need for UDI's. The AVITI24 can also perform direct in sample sequencing which includes spatial imaging of RNA, proteins, phospho-proteins, and cell morphology using [https://www.elementbiosciences.com/products/aviti24/cytoprofiling/ Teton chemistry] for cytoprofiling.
<!-- hidden
== Cytoprofiling with AVITI24 ==
[[Image:DISS_example.jpg|thumb|right|200px|Example of Teton imaging from Element Biosciences website]]
A user can optimize the assay before attempting a Teton Run. This test will require an optimization kit. Optimization is similar to creating a slide for a Teton run. The standard steps of coating (or not) that slide, loading and culturing cells prior to fixing are all similar. Instead of running the optimization slide on the AVITI24, the optimization slide should be inspected with a fluorescence objective. This process requires a separate kit from the actual slide kits.
[[Image:OptimizationSlide.jpg|thumb|middle|Lamason Lab Slide provided from Element Biosciences]]
Users can pick up a 1- or 12-well Teton Slide kit from the Center. Currently, Poly-L-Lysine-coated and uncoated slide kits are available. Uncoated slides must be coated as laid out in the protocol and users should ensure that cell fixation is ideal for one of the possible coatings which are not PLL (currently: Collagen, Fibronectin, Gelatin, Laminin, Matrigel). Cells are loaded and cultured for the desired amount of time on coated slides. Cells are fixed and the Teton Slide may be stored at 2-8C for up to 30 days or may be run immediately on the AVITI24.
For a Teton Run, cells are washed in the appropriate Cell Paint reagents for a fixed panel or custom panel cytoprofiling assay. A single Teton slide can be used to infer Morphology, RNA within the cells, Protein and also direct in situ sequencing up to 100nt which is not robust enough for transcriptomics, but is sufficient for investigating CRISPR or TCR or other short sequences of interest.
-->

Latest revision as of 02:34, 20 June 2025

Element Sequencing[edit]

The Center currently hosts an AVITI24 platform from Element Biosciences. AVITI24 platform supports both sequencing and cytoprofiling. For most sequencing needs, the Center will use Element's Freestyle chemistry kits.

Service Element Sequencing
SUBMISSION Illumina OR Element libraries
MIN VOLUME 15 uL
MIN CONCENTRATION 2 nM
INCLUDED SERVICES
  • Quality Control:Fragment Analyzer and qPCR
  • Element Sequencing
  • Demultiplexing
ADDITIONAL SERVICES
  • Quality Control
  • Element library preperation
DATA FORMATS
  • FASTQ (stored 1 year)
QUALITY CONTROL
  • FASTQC
  • Basic run metrics (alignment rate, complexity)
  • Basic RNAseq metrics (where applicable)
  • Basic paired end metrics (where applicable)
  • Contamination checks
PRICING LINK
SUBMISSION

P5/P7 anchored library (Illumina) is preferred. Element Adept libraries (SP5/SP27) require a 5'-phosphate on the forward end in order to circularize and sequence. Compared to an Illumina platform, AVITI24 reads are similar in read direction to Illumina's Forward Strand workflow for Dual Indexed Libraries.

Minimum reads per lane are guaranteed if:

  • the BMC has performed quality control,
  • the samples have high diversity, especially within the first 5 bases of read 1, and
  • submitted libraries are at least 2 nM.

Lane-by-lane Notes[edit]

  • The wait is variable
  • Custom primers must be compatible with TruSeq, Nextera, TruSeq, smRNA and Element sequencing primers

All other requests require a full flowcell. 300PE kits only have a single lane.

  • Min custom primer submission: 70 uL at 100 uM for Read 1, all other reads require 40 uL at 100 uM.

The AVITI24 Platform[edit]

AVITI24 NOTES
SEQUENCER
READS/FLOWCELL
Low number is minimum per lane for standard P5/P7 libraries.
  • 250M HQ read pairs (150 for PE300)
RUN TIME
  • 75 PE 24 hours
  • 150 PE 38 hours
  • 300 PE 60 hours
KITS AVAILABLE
  • 2x75 Cloudbreak FS High
  • 2x150 Cloudbreak FS High
  • 2x300 Cloudbreak FS High
 - Single end supported
- each kit comes with 34 additional cycles
KEY NOTES
  • 4-color chemistry
  • Random flow cell
  • 2-lanes per flow cell (1 for 300PE)
  • Q40+ > 80%
  • Low-binding surface chemistry
THANKS TO
  • Element Biosciences / Scott Ritterbush


The AVITI24 can run 2 flow cells per run, each hosting 2 independently-addressable lanes. Both lanes are washed with the same reagents. Element currently offers kits with 150 and 300 cycles, both supporting 2 lanes, as well as a 600 cycle kit that supports a single lane. Index hopping rates are similar to non-patterned Illumina, limiting the need for UDI's. The AVITI24 can also perform direct in sample sequencing which includes spatial imaging of RNA, proteins, phospho-proteins, and cell morphology using Teton chemistry for cytoprofiling.


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