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: @@
 == 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.
- {| class="wikitable" border=1
+{| class="wikitable" border=1
    !width=100| Service
    !width=250| Singular Sequencing
@@ Line 40: / Line 40: @@
    |-
   |}
+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>
 == The G4 Platform ==
@@ Line 45: / Line 67: @@
   !width=100| SPEC
   !width=250| G4
- !GUARANTEES
   |-
   |'''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 ||  '''Minimum reads per lane are guaranteed if:
+* F3 per Lane: 50-100 M
-*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.'''
-=== 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 Singular sequencing. <br> Singular library fragment size is verified via [https://openwetware.org/wiki/BioMicroCenter:QC AATI's Fragment Analyzer]. <br> Singular library is quantified via qPCR.
-===Requirements===
-<OL>
-<LI>All 4 bases (A, G, C, T) must appear within the first 10 cycles of read 1.
-<LI>Lane-by-lane requests must be 50 or 150 paired-end and compatible with TruSeq, Nextera, TruSeq, smRNA and Solexa sequencing primers.  All other requests will require a 4 lane minimum since all lanes use the flowcell's sequencing primers and this includes Illumina libraries which have been converted.
-<LI>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.
   |-
 |'''RUN TIME'''||