Quantification of Bacterial Whole-Genome Sequencing Libraries for Illumina Sequencing

January 15, 2020

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

New England Biolabs

Product Name:

NEBNext Library Quant Kit for Illumina

Catalog Number:

E7630S

Accurate quantification of whole genome sequencing libraries for Illumina sequencing very critcal step for successful cluster generation and optimal sequencing output. I used NEBNext Library Quant Kit for Illumina from New England Biolabs to quantify my bacterial whole genome sequencing libraries of insert size 600 bp. This kit is a qPCR based quantification kit and just needs two primers (P5 and P7 sequences of adapters). Using this kit, I have quantified bacterial whole genome sequencing libraries to 4 nM concentration. Then 20 pM libraries were loaded onto Illumina Miseq platform sequencing and yielded best cluster generation and good sequencing output with good Q score of the bases (Q >30 for 85 % total reads).

Experimental Design and Results Summary

Application

Accurate quantification of bacterial whole genome sequencing libraries for Illumina sequencing

Starting Material

Bacterial whole genome sequencing libraries of 1/100, 1/1000 and 1/100000 diluted samples (from 4 nM estimated quantity based on Qubit assay)

Tips

None

Results Summary

Whole genome sequencing libraries were prepared after shearing (to 500 bp), adaptor ligation and introduction of indexing sequences through PCR. Then these were quantified by qubit based assay and diluted to 1/100, 1/10000 and 1/100000 fold and used for quantification using NEBNext Library Quant Kit according to kit's instructions using primers (P5 and P7 primers). Based on Ct values of the samples been extrapolated from standard curve, my libraries were found to be 4 nM concentration. Then these libraries were diluted to 20 pM and used for sequencing in Illumina Miseq.

DOI or PMID #

N/A

Image Gallery

Summary

The Good

Less hands-on time for setting up the assay (usually less than an hour). Needs only two primers for all types of libraries and single extension time.

The Bad

None

The Bottom Line

This kit is a must for accurate quantification of whole genome sequencing libraries to be sequenced in Illumina sequencing platforms.

Join the discussion