Preparation of long read nucleic acid libraries

A method for preparing a nucleic acid library, comprising:

• (a) obtaining a plurality of transposomes comprising transposon adaptors, wherein the plurality of transposomes is immobilized on a plurality of beads;
• (b) contacting a plurality of nucleic acid fragments with the plurality of transposomes to obtain a plurality of polynucleotides;
• (c) amplifying the plurality of polynucleotides to obtain amplified polynucleotides by:
• (i) performing a mutagenesis PCR, such that mutations are introduced into amplified polynucleotides, and
• (ii) performing a suppression PCR;
• and
• (d) adding library adapters to each end of the amplified polynucleotides, thereby obtaining the nucleic acid library;
• further comprising enriching for target polynucleotides in (i) the plurality of polynucleotides, (ii) the amplified polynucleotides, and/or (iii) nucleic acids of the nucleic acid library by hybridizing a plurality of selection probes to the target polynucleotides;
• wherein the selection probes of the plurality of selection probes comprise at least 50 different nucleotide sequences;
• wherein each selection probe of the plurality of selection probes comprises a nucleotide sequence capable of hybridizing to a gene selected from:
• (i) TABLE 1B, (ii) a nucleotide sequence within no more than 10 kbp 5′ or no more than 10 kbp 3′ of a gene in a human genome selected from TABLE 1B, and/or (iii) a cytochrome P450 gene, an HLA gene, or a UDP-glucuronosyltransferase gene.

A kit comprising:

• a first bead-linked transposomes (BLT-1) reagent, wherein the BLT-1 transposomes reagent comprises a first adaptor sequence;
• a mutagenesis reagent comprising a first primer, dPTPs, dNTPs, and a polymerase;
• a second bead-linked transposomes (BLT-2) reagent, wherein the BLT-2 transposomes reagent comprise the first adaptor and a second adaptor;
• an amplification reagent comprising a first primer, a second primer, dNTPs, and a polymerase;
• and
• a plurality of selection probes, wherein each selection probe of the plurality of selection probes comprises a nucleotide sequence capable of hybridizing to a gene selected from:
• (i) TABLE 1B, (ii) a nucleotide sequence within no more than 10 kbp 5′ or no more than 10 kbp 3′ of a gene in a human genome selected from TABLE 1B, and/or (iii) a cytochrome P450 gene, an HLA gene, or a UDP-glucuronosyltransferase gene;
• wherein BLT-1 has a lower transposome density as compared to BLT-2;
• wherein the first primer hybridizes to the first adaptor sequence and the second primer hybridizes to the second adaptor sequence.

A system for preparing a nucleic acid library, comprising:

• (a) a first plurality of transposomes comprising transposon adaptors for tagmenting a plurality of nucleic acid fragments, wherein the first plurality of transposomes is immobilized on a first plurality of beads at a first density;
• (b) reagents for amplifying the plurality of polynucleotides to obtain amplified polynucleotides, wherein the amplifying comprising a mutagenesis PCR and/or a suppression PCR, wherein:
• (i) the first reagent for performing mutagenesis PCR comprise a low bias DNA polymerase and/or a nucleotide analogue, and
• (ii) the first reagents for performing suppression PCR comprise amplification primers having the same nucleotide sequence capable of hybridizing to the transposon adaptors;
• (c) a plurality of selection probes for enriching for target polynucleotides in the amplified polynucleotides, wherein each selection probe of the plurality of selection probes comprises a nucleotide sequence capable of hybridizing to a gene selected from:
• (i) TABLE 1B, (ii) a nucleotide sequence within no more than 10 kbp 5′ or no more than 10 kbp 3′ of a gene in a human genome selected from TABLE 1B, and/or (iii) a cytochrome P450 gene, an HLA gene, or a UDP-glucuronosyltransferase gene;
• and
• (d) a second plurality of transposomes comprising library adaptors for adding library adaptors to each end of the amplified polynucleotides, wherein the second plurality of transposomes is immobilized on a second plurality of beads at a second density, wherein the first density is less than the second density.