微量测序新方法2015
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Direct Sequencing from the Minimal Number of DNA Molecules Needed to Fill a 454 Picotiterplate
PLOS ONE 2014
454 sequencing platforms:
Starting material for a rapid library in 454 FLX + technology: 1ug
The most commonly used method to increase the initial amount of DNA for sequencing: Multiple Displacement Amplification (MDA)
MDA
Bias:
Genome coverage bias.
E. coli Genome Mapping
The ቤተ መጻሕፍቲ ባይዱS sample covered a greater part of the reference genome (47.43%) than the MDA sample (2.45%).
Moreover, only 2.10% of the sequences
However, quite often the amount of DNA available is limited: biopsies, laser dissection experiments, genomics for non-cultivable microorganisms, single cell genomic experiments, etc.
In addition, regions of high GC content could lead
to amplification biases. The low specificity of the random hexamers together with an amplification temperature of 30℃ make the MDA reaction prone to amplifying template-free hexamer concatenations, to be contaminated by alien sequences, and to the formation of chimeric sequences. Inaccurate quantification of CNVs [1]; Failure to detect both alleles, leading to
Library Preparation
DSsample-Y3: 414,443 DSsample-Y5: 41,043 经4cycles PCR 170,000 DSsample-Y3: 5,773,461 DSsample-Y5: 384,561
Sequencing Results
There is a significant decrease in GC content in the MDAsample (46.10%) compared to the DSsample (48.74%, t-test, p-value = 0.0021).
required to fill the target picotiterplate (PTP) region. We replaced the steps in
which DNA is lost in the standard 454 protocol by more sparing alternatives and
Library Preparation
heterozygous loci being miscalled as homozygous [1].
[1] Single-Cell Sequencing Technologies: Current and Future. Journal of Genetics and Genomics 41 (2014) 513-528
Objective:
The objective of this work is to obtain a 454 shotgun library for DS starting from
the amount of DNA needed to reach exactly the minimal number of molecules
of the MDAsample matched the E. coli
genome, whereas this figure was 80.59% for DSsample
E. coli Genome Mapping
P=0.0017；DS 高于MDA 15倍
最大 121× 平均 0.05×
最大 15× 平均 0.76×
quantified these minimal libraries with the qPCR assay designed by Zheng and collaborators. To assess whether DS can be proposed as an alternative to MDA we compared the sequencing results obtained using both methods.
PLOS ONE 2014
454 sequencing platforms:
Starting material for a rapid library in 454 FLX + technology: 1ug
The most commonly used method to increase the initial amount of DNA for sequencing: Multiple Displacement Amplification (MDA)
MDA
Bias:
Genome coverage bias.
E. coli Genome Mapping
The ቤተ መጻሕፍቲ ባይዱS sample covered a greater part of the reference genome (47.43%) than the MDA sample (2.45%).
Moreover, only 2.10% of the sequences
However, quite often the amount of DNA available is limited: biopsies, laser dissection experiments, genomics for non-cultivable microorganisms, single cell genomic experiments, etc.
In addition, regions of high GC content could lead
to amplification biases. The low specificity of the random hexamers together with an amplification temperature of 30℃ make the MDA reaction prone to amplifying template-free hexamer concatenations, to be contaminated by alien sequences, and to the formation of chimeric sequences. Inaccurate quantification of CNVs [1]; Failure to detect both alleles, leading to
Library Preparation
DSsample-Y3: 414,443 DSsample-Y5: 41,043 经4cycles PCR 170,000 DSsample-Y3: 5,773,461 DSsample-Y5: 384,561
Sequencing Results
There is a significant decrease in GC content in the MDAsample (46.10%) compared to the DSsample (48.74%, t-test, p-value = 0.0021).
required to fill the target picotiterplate (PTP) region. We replaced the steps in
which DNA is lost in the standard 454 protocol by more sparing alternatives and
Library Preparation
heterozygous loci being miscalled as homozygous [1].
[1] Single-Cell Sequencing Technologies: Current and Future. Journal of Genetics and Genomics 41 (2014) 513-528
Objective:
The objective of this work is to obtain a 454 shotgun library for DS starting from
the amount of DNA needed to reach exactly the minimal number of molecules
of the MDAsample matched the E. coli
genome, whereas this figure was 80.59% for DSsample
E. coli Genome Mapping
P=0.0017；DS 高于MDA 15倍
最大 121× 平均 0.05×
最大 15× 平均 0.76×
quantified these minimal libraries with the qPCR assay designed by Zheng and collaborators. To assess whether DS can be proposed as an alternative to MDA we compared the sequencing results obtained using both methods.