BioMicroCenter:Singular Sequencing: Difference between revisions

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	MIT - ilabs

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

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

@@ Line 1: / Line 1: @@
-[[Image:G4-open.png|right|900px]]<br>
 == Singular Sequencing ==
-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>
+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.
-=== Workflow ===
+{| class="wikitable" border=1
-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.'''
+  !width=100| Service
-===Requirements/Things to Consider===
+  !width=250| Singular Sequencing
-<OL>
+  |-
-<LI>All 4 bases (A, G, C, T) must appear within the first 10 cycles of read 1.
+  |INPUT || Singular libraries
-<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.
+  |-
-<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.
+  |MIN VOLUME || 12 uL
-<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.
+  |-
-<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.
+  |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
+  |
+* [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#SINGULAR_G4|LINK]]
+  |-
+  |SUBMISSION
+  |
+* MIT - [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=3863 ilabs]
+  |-
+ |}
+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===
+*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
+[[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 ===
-=== Illumina Library to Singular Library Conversions and Custom Sequencing ===
-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.
 == The G4 Platform ==
@@ Line 23: / Line 70: @@
   |'''SEQUENCER'''||[[Image:2023_Singular_G4.jpg|center|200px]]
   |-
-  | '''READS/LANE'''<BR> Low number is minimum per lane for standard Illumina libraries.||
+  | '''READS/LANE'''<BR> Low number is minimum per lane for standard libraries||
 * F3 per Lane: 50-100 M
   |-
 |'''RUN TIME'''||