Next-Generation Sequencing Core
http://ngsc.med.upenn.edu
jschug@upenn.edu
The NGSC offers high-throughput sequencing services from experimental design, sample
quality checks, library preparation, sequencing, and basic data analysis.
Sequencing
NEW! We are purchasing a new hiSeq2500 and a NextSeq500.
hiSeq2000 High output sequencing from 50bp single read (SR) to 100bp paired-end sequencing (PE).
hiSeq2500 Rapid sequencing up to 150PE.
miSeq Very rapid sequencing, lower read counts but up to 300PE sequencing. Best for metagenomics or other small-scale targeted sequencing, miRNA-seq for small sample counts, or
library testing.
NextSeq Very rapid sequencing, up to 150PE. Medium or high-output. Should be available in 2015.
Library Prep
We can prepare RNA-Seq, smRNA-Seq, ChIP-Seq, Whole-Genome libraries for you. RNA-Seq libraries can be
made for half or whole 96-well plates.
Contact Olga Smirnova (smirnovamail.med.upenn.edu) for technical details.
High-throughput Library Prep
The NGSC has the following equipment to help with high-throughput library preparations.
Agilent Bravo Liqof ultrasound is delivered to up to 8 samples at a time for
uid Handler NEW!
rapid processing. Also suitable for ChIP-Seq chromatin
sonication and extracting DNA from FFPE samples.
The Agilent Bravo liquid handler is programmed with protoFluidigm C1 - Sincols for exome-capture Images/NGS-Automation-Set-Up.jpg
gle cell RNA-Seq
libraries as well as AgiStarting with 1000 cells
lent’s RNA-Seq library prep kit. Other protocols are be in 5ul, the C1 can
available as well.
capture up to 96 cells
in a microfluidics chip.
Cells in the chip can Images/c1_left_250x225.jpg
Covaris
LE220
be examined with a miUltrasonicator
NEW! The Covaris
croscope and annotated
sonicator does not use a
for later. The chip is returned to the C1 for lyprobe to deliver power
sis, RNA extraction, and cDNA synthesis. The next day
to the sample, so there
Images/le220_md.jpg
is no risk of contaminaIllumina’s Nextera XT kit is used to make libraries from
5 to 96 samples.
tion. A focussed beam
Data Analysis
The NGSC offers basic data analysis of many experiment types. We are coordinating with the IBI Bioinformatics
Core (http://ibi.upenn.edu/bi-core/) on data sharing and a wider set of more advanced analysis pipelines.
We deliver data via the web or to your account on PMACS.
C1
C1-capture
C1-extraction
C1-IFC-dna
C1-IFC-mrna
C1-reagents-dna
505
410
910
780
1020
plate
plate
plate
plate
plate
C1-reagents-mrna
685
plate
qc-bioAnalyzer
15
sample
qc-Kapa
45
sample
qc-Pooling
15
sample
qc-qubit
5
sample
qc-qubit-nextera
5
sample
qc-tapeStation
5
sample
LP-ChIP-Seq/NEBnext
60
sample
LP-DNA-Seq/Exome-SSCR
465
sample
LP-DNA-Seq/truSeq-noAmp
40
sample
LP-NexteraXT/LT-C1
65
sample
LP-RNA-Seq/Clontech-LowInput
170
sample
LP-RNA-Seq/NugenOvationAmplification
245
sample
LP-RNA-Seq/riboZeroANDtruSeqStranded
190
sample
LP-RNA-Seq/truSeq
105
sample
LP-RNA-Seq/truSeqStranded
115
sample
LP-smRNA-truSeq
150
sample
LP-NexteraXT/WholePlate
4060
plate
LP-NexteraXT/WholePlate-C1
1815
plate
LP-RNA-Seq/truSeqStrandedPolyA/HalfPlate
5170
plate
LP-RNA-Seq/truSeqStrandedPolyA/WholePlate
9795
plate
LP-RNA-Seq/truSeqStrandedRibo/HalfPlate
6550
plate
LP-RNA-Seq/truSeqStrandedRibo/WholePlate
12555
plate
sq-050PE-hiSeq-V3
1435
lane
sq-050PE-hiSeq-V4
1665
lane
sq-050PE-hiSeqRapid-V2
2005
lane
sq-050SR-hiSeq-V3
905
lane
sq-050SR-hiSeq-V4
1040
lane
sq-050SR-hiSeqRapid-V2
1365
lane
capture on C1, not including reagents
post capture phase of C1, ether RNA/cDNA or WGA
Fluidigm IFC for DNA extraction and WGA - any cell size
Fluidigm IFC for RNA extraction and cDNA - any cell size
Fluidigm reagents for DNA extraction and WGA - any cell
size
Fluidigm reagents for RNA extraction and cDNA - any cell
size
QualityCheck
RNA or library quality check with Agilent bioAnalyzer using any of the DNA or RNA chips.
Molarity measurement of library using Kapa Biosystems
Library quant kit. 3 technical replicates of 3 dilutions of
sample.
Mix samples in equi-molar amounts to ensure as close to
even read counts as possible.
RNA or DNA concentration measurement using LifeTech
qubit fluorimeter.
Kapa-based assessment of a large batch of samples from
NexteraXT library prep. Deprecated
RNA or library quality check with Agilent TapeStation meant for large sample batches.
LibraryPrep-LT
Low-throughput library prep for ChIP-Seq samples (or
other dsDNA samples) using the NEBNext ChIP-Seq kits.
Labor extra.
Library prep from gDNA of human exome libary using the
Agilent SureSelect Clinical Research Exome kit
Low-throughput library prep for DNA-Seq samples using
the Illumina TruSeq DNA PCR kit. Labor extra.
Low-throughput library prep using Nextera-XT kit on a
small number of C1 cDNA samples. Labor included.
Generation of cDNA from small amounts of total RNA using the Clontech SMARTer Ultra Low Input RNA kit. Labor extra.
Generation of cDNA from small amounts of total RNA using the NuGen Ovation kit. Labor extra.
Low-throughput library prep for RNA-Seq samples using Illumina TruSeq Stranded Total RNA with Ribo Zero
Gold Set. Labor extra.
Low-throughput library prep for RNA-Seq samples using
the older non-stranded Illumina kits. Labor extra. Deprecated.
Low-throughput library prep using Illumina truSeq
stranded mRNA kit. Labor extra.
Low-throughput library prep using Illumina small RNA kit
- i.e., miR-Seq. Labor extra.
LibraryPrep-HT
High-throughput library prep using Nextera-XT in most of
a 96-well plate of DNA samples. Labor included.
High-throughput library prep using Nextera-XT for C1
samples in most of a 96-well plate of DNA samples. Labor included.
High-throughput library prep using Illumina truSeq
stranded mRNA kit for nearly half of a 96-well plate. Labor
included.
High-throughput library prep using Illumina truSeq
stranded mRNA kit for most of a 96-well plate. Labor
included.
High-throughput library prep using Illumina truSeq
stranded total RNA (ribo-Zero) kit for nearly half of a 96well plate. Labor included.
High-throughput library prep using Illumina truSeq
stranded total RNA (ribo-Zero) kit for most of a 96-well
plate. Labor included.
Sequencing
Sequencing 2 ends, 50bp each, on HiSeq in high-output
mode to get about 180 million reads or pairs
Sequencing 2 ends, 50bp each, on HiSeq in high-output
mode to get about 250 million reads or pairs
Sequencing 2 ends, 50bp each, on HiSeq in rapid mode to
get about 150 million reads or pairs
Sequencing 1 ends, 50bp each, on HiSeq in high-output
mode to get about 180 million reads or pairs
Sequencing 1 ends, 50bp each, on HiSeq in high-output
mode to get about 250 million reads or pairs
3Sequencing 1 ends, 50bp each, on HiSeq in rapid mode to