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== Singular Sequencing ==
  |- style="vertical-align: top;"
The Center currently hosts a G4 platform by [https://singulargenomics.com/| Singular Genomics]. Singular’s G4 platform is a short-read mid-throughput sequencing platform delivering adequate sequencing data through its proprietary sequencing-by-synthesis chemistry at a significantly competitive price point.  
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{| class="wikitable" border=1
{| class="wikitable" border=1
   !width=100| Service  
   !Service  
   !width=250| Singular Sequencing
   !Singular Sequencing
   |-
   |-
   |INPUT || Illumina or Singular libraries
   |INPUT || Singular libraries  
   |-
   |-
   |MIN VOLUME || 12 uL
   |MIN VOLUME || 12 uL
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   |ADDITIONAL SERVICES ||
   |ADDITIONAL SERVICES ||
* Quality Control
* Quality Control
* Singular library preperation
* Singular library preparation
   |-
   |-
   |DATA FORMATS  
   |DATA FORMATS  
   |
   |
*FASTQ (stored 1 year)
*FASTQ (stored 1 year)
*BCL (stored 30d)
   |-
   |-
   |QUALITY CONTROL  
   |QUALITY CONTROL  
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  |}
  |}


== Singular Sequencing ==
Compared to a typical Illumina library, a Singular index 1 would be the reverse compliment of the i5 index, whereas index 2 would be the reverse compliment of the i7 index.
The Center currently hosts a G4 platform by [https://singulargenomics.com/| Singular Genomics].  Singular’s G4 platform is a short-read mid-throughput sequencing platform delivering adequate sequencing data through its proprietary sequencing-by-synthesis chemistry at a significantly competitive price point.  The G4 platform is unique in its flexibility to run 1 to 4 flow cells in parallel, each with 4 independent lanes.<br>
 
=== Workflow ===
===Lane-by-lane Requirements===
Library prep options for [https://openwetware.org/wiki/BioMicroCenter:DNA_LIB DNA] or [https://openwetware.org/wiki/BioMicroCenter:RNA_LIB RNA] are available for Singular sequencing. Once Singular-ready or Illumina 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], adapt Singular flow cell binding sites S1/S2 if needed, and the quantify the final loading pool via qPCR.  '''We can only guarantee minimum reads per lane if: a)the BMC has performed quality control, b)the samples are high-complexity, especially for the first 10 nt of read 1, and c)submitted libraries are at least 2 nM.'''
*Must be 50 or 150 paired-end dual indexed
===Requirements/Things to Consider===
*Pool must be compatible with TruSeq, Nextera, TruSeq, smRNA and Solexa sequencing primers
<OL>
 
<LI>All 4 bases (A, G, C, T) must appear within the first 10 cycles of read 1.
Minimum reads per lane are guaranteed if:  
<LI>Both forward and reverse read strands are present and accessible during sequencing. After the desired number of cycles have been reached for a given read, a block is introduced to prevent any continued extension of the growing read chain, but the generated read chain is NOT denatured/melted away before the priming and extension of the next read.  For this reason, lane-by-lane requests must be either 50 or 150 paired-end compatible with a dual index of 8 nt each.  All other requests will require a 4 lane (or 1 flow cell) minimum.
*the BMC has performed quality control,  
<LI>Indexes are read off flow cell binding regions, S1 and S2. For standard lane-by-lane requests, the first index will be read off the S1 region of the final library, and index 2 will be read off the S2 region. Compared to a typical Illumina library, a Singular index 1 would be the reverse compliment of the i5 index, whereas index 2 would be the reverse compliment of the i7 index.
*the samples are high-complexity, especially for the first 10 nt of read 1, and
<LI>Each flow cell runs off a single reagent cartridge, thus each lane on that flow cell will be treated with the same primer cocktail by run. For standard lane-by-lane requests, this means all custom primers must be compatible with TruSeq, Nextera, TruSeq smaRNA, and Solexa sequencing primers.
*submitted libraries are at least 2 nM.
<LI>Singular also supports the conversion of Illumina libraries through either index replacement or anchor expansion for completed libraries that wish to be run on this platform.  Index hopping rates are near zero, limiting the need for UDI's.  Early quality metrics can be seen below.
 
All other requests, including use of custom sequencing primers require a full flowcell. Illumina libraries which have been converted in the Center will also require a full flowcell.
 
*Min custom primer submission: 20 uL at 100 uM
 
 
[[IMAGE:IndexhopF3.jpeg|left|250px|Index hopping for a standard set of Singular libraries]] [[IMAGE:PPPPF3.jpeg|right|200px|Percent Perfect Plot for Singular libraries 150PE]]
 
Index hopping on Singular runs has been low. The performance of an F3 flowcell mimics other short-read 4 color chemistry sequencers with patterned flowcells.  
<br><br><br><br>
 


=== Data Handling ===
== The G4 Platform ==
=== Illumina Library to Singular Library Conversions and Custom Sequencing ===
{| class="wikitable" border=1
The BioMicro Center can modify Illumina ready libraries by using the p5 and p7 flow cell binding sequences to attach Singular S1 and S2 flow cell binding sequences.  However, this process leaves behind a 16 bp portion of the p5/p7 sequences requiring a different set of primers that will compete with the onboard S1 and S2 index primers.  Like custom runs, these requests are rare and must be run on an entire flow cell.
!width=100| SPEC
!width=250| G4
|-
|'''SEQUENCER'''||[[Image:2023_Singular_G4.jpg|center|200px]]
|-
| '''READS/LANE'''<BR> Low number is minimum per lane for standard libraries||
* F3 per Lane: 50-100 M
|-
|'''RUN TIME'''||
* 50 PE 11 hours
* 150 PE 24 hours
|-
|'''KITS AVAILABLE'''||
* 100nt
* 300nt
|-
|'''KNOWN APPLICATIONS'''||
* RNAseq including single cell
* Gene Expression
* Exome
* Target Enrichment
* Metagenomics
|-
|'''KEY NOTES'''||
* 4-color chemistry
* Patterned flow cell
* 4-lanes per flow cell
* 4-flow cells per run
* Index replacement or anchor expansion of Illumina libraries
|-
|'''THANKS TO'''||
* Singular Genomics
|}

Latest revision as of 17:26, 9 June 2025

Singular Sequencing[edit]

The Center currently hosts a G4 platform by Singular Genomics. Singular’s G4 platform is a short-read mid-throughput sequencing platform delivering adequate sequencing data through its proprietary sequencing-by-synthesis chemistry at a significantly competitive price point.

Service Singular Sequencing
INPUT Singular libraries
MIN VOLUME 12 uL
MIN CONCENTRATION 4 nM
INCLUDED SERVICES
  • Quality Control:Fragment Analyzer and qPCR
  • Singular Sequencing
  • Demultiplexing
ADDITIONAL SERVICES
  • Quality Control
  • Singular library preparation
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

Compared to a typical Illumina library, a Singular index 1 would be the reverse compliment of the i5 index, whereas index 2 would be the reverse compliment of the i7 index.

Lane-by-lane Requirements[edit]

  • Must be 50 or 150 paired-end dual indexed
  • Pool must be compatible with TruSeq, Nextera, TruSeq, smRNA and Solexa sequencing primers

Minimum reads per lane are guaranteed if:

  • the BMC has performed quality control,
  • the samples are high-complexity, especially for the first 10 nt of read 1, and
  • submitted libraries are at least 2 nM.

All other requests, including use of custom sequencing primers require a full flowcell. Illumina libraries which have been converted in the Center will also require a full flowcell.

  • Min custom primer submission: 20 uL at 100 uM


Index hopping for a standard set of Singular libraries
Index hopping for a standard set of Singular libraries
Percent Perfect Plot for Singular libraries 150PE
Percent Perfect Plot for Singular libraries 150PE

Index hopping on Singular runs has been low. The performance of an F3 flowcell mimics other short-read 4 color chemistry sequencers with patterned flowcells.




The G4 Platform[edit]

SPEC G4
SEQUENCER
READS/LANE
Low number is minimum per lane for standard libraries
  • F3 per Lane: 50-100 M
RUN TIME
  • 50 PE 11 hours
  • 150 PE 24 hours
KITS AVAILABLE
  • 100nt
  • 300nt
KNOWN APPLICATIONS
  • RNAseq including single cell
  • Gene Expression
  • Exome
  • Target Enrichment
  • Metagenomics
KEY NOTES
  • 4-color chemistry
  • Patterned flow cell
  • 4-lanes per flow cell
  • 4-flow cells per run
  • Index replacement or anchor expansion of Illumina libraries
THANKS TO
  • Singular Genomics
Retrieved from "http://bmcwiki.mit.edu/index.php?title=BioMicroCenter:Singular_Sequencing&oldid=696"