Library QC Explained
Accurate quantification and quality check (QC) of Illumina sequencing libraries is the key to a good sequencing run. At the FGCZ we use 4 different methods to determine the quality of sequencing libraries depending on the library preparation protocol and sequencing instrument.
For best sequencing results, the values we derive from qPCR, Tapestation and Qubit should be very similar.
Using qPCR, we can determine how much of the libraries contain the P5 and P7 adapters. Only fragments containing these sequences can cluster in the flowcell.
Library quantification is performed by amplifying the dilution series of library standards and diluted library samples by qPCR using primers targeting the Illumina P5 and P7 oligo sequences. The concentrations of the diluted library samples are determined by calculating against the standard curve using absolute quantification.
Using tapestation, we can determine the fragment size of the library. This is necessary because only fragments between 100bp- 800bp cluster efficiently. We also check for primer dimers.
10% of primer-dimers present in the library can result to 90% sequencing reads lost to primer dimers.
We quantify the region between 100bp to 800 bp and make sure the primer dimers are less than 3%.
Using Qubit, we can determine the total amount of DNA in the library.
We do a shallow sequencing run using the Iseq. Ultimately, this is the only way to know if the libraries will sequence well, if the pooling is equimolar and if the barcodes assigned to each sample is correct.
Accurate quantification and quality check (QC) of Illumina sequencing libraries is the key to a good sequencing run. At the FGCZ we use 4 different methods to determine the quality of sequencing libraries depending on the library preparation protocol and sequencing instrument.
For best sequencing results, the values we derive from qPCR, Tapestation and Qubit should be very similar.
QPCR
Using qPCR, we can determine how much of the libraries contain the P5 and P7 adapters. Only fragments containing these sequences can cluster in the flowcell.
Library quantification is performed by amplifying the dilution series of library standards and diluted library samples by qPCR using primers targeting the Illumina P5 and P7 oligo sequences. The concentrations of the diluted library samples are determined by calculating against the standard curve using absolute quantification.
Tapestation
Using tapestation, we can determine the fragment size of the library. This is necessary because only fragments between 100bp- 800bp cluster efficiently. We also check for primer dimers.
10% of primer-dimers present in the library can result to 90% sequencing reads lost to primer dimers.
We quantify the region between 100bp to 800 bp and make sure the primer dimers are less than 3%.
Qubit
Using Qubit, we can determine the total amount of DNA in the library.
Iseq QC
We do a shallow sequencing run using the Iseq. Ultimately, this is the only way to know if the libraries will sequence well, if the pooling is equimolar and if the barcodes assigned to each sample is correct.