Amplicon Library Preparation
Amplicon Library Preparation - Related Videos
08:57
Amplicon Sequencing using the Long-Read Sequencing Technologies
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2025
This protocol was optimized for targeted deep sequencing of 18 drug-resistance regions in Mycobacterium tuberculosis using a long-read sequencing platform, followed by analysis with a tuberculosis-specific bioinformatics pipeline designed for long-read data.
12:01
3' End Sequencing Library Preparation with A-seq2
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Cited by 9 •
2017
This protocol describes a method for mapping pre-mRNA 3' end processing sites.
10:24
Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
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Cited by 119 •
2014
Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.
09:12
MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells
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Cited by 11 •
2011
Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.
08:37
Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos
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Cited by 1 •
2020
We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.
FAQs
Primer design determines which DNA region is amplified and how consistently different templates enter the library. Primers must target the defined locus appropriately, because poor selection can exclude relevant sequences or favor some templates over others. These amplification biases may distort estimates of genetic variation or microbial composition, making primer choice central to representative and interpretable results.
Adapters make amplified DNA products compatible with the sequencing workflow, while sample-specific indexes provide identifying tags. Indexed products can therefore be pooled for sequencing and assigned back to their source samples during analysis. This organization supports efficient comparison of multiple samples while preserving the connection between each sequence result and its original biological material.
Targeted enrichment concentrates sequencing effort on selected DNA regions rather than distributing it across an entire genome. Amplicon Library Preparation consequently supports focused investigation of loci relevant to a study, such as genotype variation or microbial markers, with less sequencing capacity than whole-genome approaches. The tradeoff is that conclusions remain limited to the amplified regions.
The workflow begins by using polymerase chain reaction to amplify the selected locus. Sequencing adapters and sample-specific indexes are then added or incorporated into the amplification products. Products from different samples can subsequently be pooled for sequencing, with indexes enabling sample identification. Each stage must preserve the intended target and minimize amplification bias.
This approach is useful when a study focuses on defined genetic markers rather than complete genomes. Supported applications include examining bacterial or viral composition, biodiversity, genotype variation, and environmental samples. By concentrating analysis on selected loci, it can provide a practical way to compare targeted biological features across samples without requiring whole-genome sequencing.
Amplification bias can make some templates appear more or less abundant than they are in the original sample. As a result, observed microbial composition, biodiversity, or genetic variation may not fully represent the starting material. Careful primer design and deliberate control of amplification bias are therefore necessary to produce results that support reliable biological interpretation.