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Comparison of SYBR enzymes and standards in Illumina library quan:fica:on Kevin Thai and Stuart Levine
MIT BioMicro Center
qRT-‐PCR: Confirm anchors and concentra9on
Success of qPCR library quan9fica9ons depend on the accuracy and reproducibility of standards used in addi9on to the ability of DNA polymerase to efficiently amplify all adaptor-‐flanked libraries. We compared the (i) Roche SYBR Green, (ii) KAPA SYBR Fast polymerases and (iii) Qiagen Quan9Fast SYBR Green and the PhiX standards and KAPA pre-‐diluted standards to determine the rela9ve importance of these two factors in library quan9fica9on by qPCR.
Objec:ve: Comparing various SYBR enzymes and DNA standards for accurate quan9fica9on of libraries generated for Illumina next-‐genera9on sequencing.
Comparison of Standards: Seven serial dilu9ons of PhiX were plated on plates 1 and 2 to test for reproducibility. A Third plate was run with pre-‐diluted KAPA DNA standards. Iden9cal Illumina libraries were loaded on all plates in triplicates and run on the Roche 480 Lightcycler. The standards were used to generate a linear curve to determine the concentra9on of each library.
Comparison of enzymes: Three plates were loaded with Illumina libraries and PhiX standards. Both plates were amplified with three different enzymes: (1) Roche SYBR Green (2) KAPA SYBR FAST (3) Qiagen Quan9Fast. Cluster counts from the Illumina sequencers were analyzed with respect to concentra9ons obtained from the enzymes.
Our results indicate that the DNA polymerase from the KAPA SYBR Fast kit is be`er suited to library quan9fica9on applica9ons. The Roche SYBR Green kit showed significant variability in respect to cluster genera9on using the obtained concentra9on from RT-‐qPCR, possibly related to sample type or insert size. Similar results were obtained with the Quan9fast SYBR Green, in that libraries with atypical fragment sizes were inaccurately quan9fied. No differences were observed between the two types of standards used, making DNA polymerase the determining factor in the successful amplifica9on of each library. This enzyme is also amenable to automated liquid handling, allowing high-‐throughput sample analysis, making it ideal for core laboratory seangs.
Gene Expression Analysis Genotyping DNA Quan9fica9on …More
SYBR
0
50000
100000
150000
200000
250000
300000
350000
400000
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
Clu
ster
s/m
m^2
Clusters (PF) on GAIIx (KAPA)
q1
min
median
max
q3
0
50000
100000
150000
200000
250000
300000
350000
400000
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
Clusters/m
m^2
Clusters (PF) on GAIIx (Roche)
q1
min
median
max
q3
HiSeq2000 GAIIx
SPRI-‐TE
Sample Prep & Quality Control
LabChip GX Roche LC480
MICROARRAYS Affymetrix Agilent
SERVICES: Affymetrix Agilent
Exon 3 prime
Prokaryote
miRNA CGH
100ng 3µg
100ng 50ng*
100ng
50ng* 50ng*
* <1ng possible in some cases
AUTOMATION Tecan EVO150
Biomark
Varioskan
-‐1
-‐0.5
0
0.5
1
1.5
2
2.5
3
DSN-‐2 Mouse C Brain A4600 JW-‐H3B U87 H 1 Alnylam 23 Crummy 4 HuGE
Log [Con
c] (n
M)
Sample
QC Results -‐ KAPA
Bioanalyzer
Roche
KAPA
-‐1.5
-‐1
-‐0.5
0
0.5
1
1.5
2
2.5
3
JW-‐H27c 106-‐1 Rhesus A Liver Y182 Total 12 tx2-‐B 7-‐Crummy 12-‐Crummy
Log [Con
c.] (nM
)
Sample
QC Results -‐ Qiagen
Bioanalyzer
KAPA
Qiagen
0
2
4
6
8
10
12
14
16
Std. 1 Std. 2 Std. 3 Std. 4 Std. 5 Std. 6 Std. 7
Standards
Concen
tra:
ons (nM)
PhiX Standards -‐ Plate 1
0
2
4
6
8
10
12
14
Std. 1 Std. 2 Std. 3 Std. 4 Std. 5 Std. 6 Std. 7
Standards
Concen
tra:
on (n
M)
KAPA Standards -‐ Plate 2
0
50000
100000
150000
200000
250000
300000
350000
1 2 3 4 5 6 7
Clusters/m
m^2
Lane
Average Clusters
Roche
KAPA
Y=-‐3.442x+12.89 Error=0.0007
*Average clusters for six consecu9ve flowcells loaded with (i) Roche-‐generated concentra9ons and (ii) KAPA-‐generated concentra9ons.
0.1
1
10
100
1000
267 285 303 337 344 347 612 702
Log [Con
c.] (nM
)
Insert Size
Concentra:on vs. Insert Size
KAPA
Roche
Efficiency of Enzymes: This data indicates that the KAPA enzymes has greater sensi9vity with larger amplicons than Roche, resul9ng in accurate quan9fica9on of Illumina libraries of various sizes.
Conclusion:
ILLUMINA SEQUENCING
