Methods for production and quantification of unique molecular identifier-labeled beads

A method for preparing a unique molecular labeled substrate, the method comprising:

• (a) providing a substrate comprising one or more oligonucleotide templates attached to the substrate,
• wherein the substrate is a bead, derivatized bead, derivatized glass slide, or derivatized polymer;
• (b) adding a primer template partially complementary to the one or more oligonucleotide templates;
• wherein:
• the primer template comprises a single stranded oligonucleotide having a structure comprising:
• 5′-Antisense-N8-20-Barcode-N8-20-Anchor-N4-20-x-3′,
• where:
• Antisense-N8-20 is an antisense sequence of 8 to 20 nucleotides complementary to an anchor sequence of the oligonucleotide template or the extended product:
• Barcode-N8-20 is a barcode region of 8 to 20 nucleotides;
• Anchor-N4-20 is an anchor sequence of 4 to 20 nucleotides;
• and
• -x is a 3′-blocking moiety to prevent extension from the 3′-terminus of the primer template;
• (c) adding reagents sufficient to perform an extension reaction;
• (d) incubating the extension reaction for a period of time sufficient to produce an extension product;
• (e) purifying the substrate comprising the extension product;
• (f) repeating steps (b) to (e) at least two additional times using additional primer templates partially complementary to the extended product from each subsequent round;
• (g) purifying the substrate comprising the final extension product using the same process as in step (e);
• and
• (h) optionally, quantitating the amount of the oligonucleotide templates attached to the substrate, the amount of extension product attached to the substrate at each round, or the amount of the final extension product attached to the substrate.