How to calculate NGS read length

What is Sequencing Read Length

What is Sequencing Read Length?

Next-generation sequencing (NGS) read length refers to the number of base pairs (bp) sequenced from a DNA fragment. After sequencing, the regions of overlap between reads are used to assemble and align the reads to a reference genome, reconstructing the full DNA sequence. Sequencing read lengths correspond directly to the sequencing reagents used on an NGS instrument—more chemistry cycles generate longer reads.

Why is NGS Read Length Important

Why is NGS Read Length Important?

Choosing the right sequencing read length depends on your sample type, application, and coverage requirements. Because long reads allow for more sequence overlap, they are useful for de novo assembly and resolving repetitive areas of the genome with greater confidence. For other applications, such as expression profiling or counting studies, shorter reads are sufficient and more cost-effective than longer ones.

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Sequencing Read Options

There are two sequencing read types: single-read and paired-end sequencing. Single-read sequencing involves sequencing DNA fragments from one end to the other. It is useful for some applications, such as small RNA sequencing, and can be a fast and economical option.

With paired-end sequencing, after a DNA fragment is read from one end, the process starts again in the other direction. In addition to producing twice the number of sequencing reads, this method enables more accurate read alignment and detection of structural rearrangements. Today, most researchers use the paired-end approach.

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Library Prep Kit Selector

This tool provides recommended read lengths for different methods and Illumina library prep kits.

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How to Calculate Illumina Read Length

All Illumina sequencing reagents feature a certain number of sequencing cycles. These cycles are directly related to sequencing read length. Because one base is sequenced per cycle, the total number of cycles indicates the maximum number of bases that can be sequenced. You can use sequencing reagents to generate single continuous reads or for paired-end sequencing in both directions. (For example, a 300-cycle kit can be used for a 1 × 300 bp single-read run or a 2 × 150 bp paired-end run.)

DNA Sequencing Applications
Application Recommended Read Length
Whole-genome sequencing 2 × 150 bp
Whole-exome sequencing 2 × 150 bp
Targeted enrichment sequencing 2 × 150 bp
Amplicon sequencing Length of the entire amplicon insert
De novo sequencing Ranges from 2 × 150 to 2 × 300 bp
RNA Sequencing Applications
Application Recommended Read Length
Transcriptome analysis 2 × 75 bp
Gene expression profiling 1 × 50 bp
Small RNA sequencing 1 × 50 bp
Beginner's Guide to Next-Generation Sequencing

Considering bringing next-generation sequencing to your lab, but unsure where to start? These resources cover key topics in NGS and are designed to help you plan your first experiment.

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Beginner's Guide to Next-Generation Sequencing
Sequencing Coverage Calculator

This tool helps you estimate sequencing coverage and contains read length recommendations for your experiment.

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NGS Read Length and Coverage

Coverage depth refers to the average number of sequencing reads that align to, or "cover," each base in your sequenced sample. The Lander/Waterman equation1 is a method for calculating coverage (C) based on your read length (L), number of reads (N), and haploid genome length (G): C = LN / G

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Considerations for RNA Sequencing Read Length

Considerations for RNA Sequencing Read Length

Different RNA-Seq experiment types have unique sequencing read length and depth requirements. This bulletin reviews read length and depth considerations and offers resources for planning experiments.

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NGS Experimental Design & Protocols

Find tips and resources to help you plan your sequencing runs.

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What is my maximum supported read length?

Depending on your sequencing platform and SBS kit version, your maximum supported read length can vary. Read the latest bulletin to learn more about your maximum read length along with other resources to optimize data quality.

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Have questions about our products or how to get started with NGS? Connect with an Illumina representative.

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  1. Lander ES, Waterman MS. Genomic mapping by fingerprinting random clones: a mathematical analysis. Genomics. 1988;2(3):231-239.