Application of a PCR sequencing method, based on DNA barcoding technique and DNA incomplete shearing strategy, in HLA genotyping

A method for determining the nucleotide sequence of a nucleic acid of interest, comprising:

• a) providing n samples, wherein n is an integer of ≥1;
• b) optionally dividing the n samples into m groups, wherein m is an integer and n≥m≥1;
• c) performing PCR amplification on the samples under conditions suitable for amplifying the nucleic acid of interest when templates from the samples are available, wherein a pair or multiple pairs of index primers are used for each sample, each pair of index primers consists of a forward index primer and a reverse index primer, and each index primer consists of a PCR primer and a primer index added to the 5′-end of the PCR primer, wherein the primer index of the forward index primer and the primer index of the reverse index primer in each pair of index primers are identical or different, and wherein the primer indexes in the pair of index primers used for different samples are different;
• d) pooling products of the PCR amplification from each of the samples together;
• e) subjecting the amplified products to incomplete shearing to obtain a mixture of intact un-sheared PCR products and partially sheared PCR products, and purifying and recovering said mixture of intact un-sheared PCR products and partially sheared PCR products;
• f) constructing a PCR-free sequencing library based on the mixture of intact un-sheared PCR products and partially sheared PCR products recovered in e), wherein different library adapters are added to distinguish different PCR-Free sequencing libraries;
• g) purifying and recovering DNA bands between the maximum read length and the maximum applicable DNA length of a sequencer using the second generation sequencing technique;
• h) subjecting the recovered DNA mixture to the sequencer to obtain sequences of the sheared DNA;
• and
• i) matching obtained sequencing data to corresponding samples based on a unique primer index for each sample, aligning obtained sequences of the sheared DNA against DNA reference sequences corresponding to the PCR products, and assembling a complete sequence of the nucleic acid of interest from the obtained sequences of the sheared DNA based on sequence overlap and linkage relationship, whereby a length of the complete sequence of the nucleic acid of interest exceeds a maximum read length of a sequencer used for said sequencing.