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Next-generation sequencing (NGS) is a massively parallel sequencing technology: instead of reading one DNA fragment at a time, an instrument reads very large numbers of fragments at once. Illumina’s technology overview describes the NGS workflow as library preparation, sequencing and data analysis, and it gives the output of a single run as ranging from 300 kilobases up to multiple terabases. The same overview notes that a run can carry hundreds to thousands of samples. This guide covers the workflow, how to read what comes out, and how to reuse public sequence data. “DNA sequencing NGS” and “NGS DNA sequencing” name the same method.
What NGS is used for in research
Because one run can carry many samples and a very large number of reads, NGS is the standard route when a project needs more data than a one-fragment-at-a-time method can economically deliver. Illumina contrasts it with the Sanger-based Human Genome Project, which took over 10 years. The NHGRI DNA Sequencing Fact Sheet (page updated June 27, 2023) states that some labs can sequence well over 100,000 billion bases per year. For a short, targeted read of one amplicon, the older method still applies; see Sanger sequencing and the broader DNA sequencing methods overview.
Access: reusing public NGS data before sequencing anything new
The fastest NGS project is sometimes one that never generates a new library. The NCBI Sequence Read Archive (SRA) is described in its documentation as the world’s largest publicly available repository of raw, unassembled genetic sequence data. SRA is mirrored with EMBL-EBI in England and NIG in Japan through the INSDC collaboration. Human data that carries access restrictions is available only through dbGaP Authorized Access. NCBI publishes a download guide at ncbi.nlm.nih.gov/sra/docs/sradownload and a submission guide at ncbi.nlm.nih.gov/sra/docs/submit. For the CASRAI view of the archive, see Sequence Read Archive (SRA); for depositing your own reads, see submitting sequence data to NCBI SRA.
Step-by-step: the four-stage NGS workflow
Illumina lists three stages (library preparation, sequencing, data analysis). Thermo Fisher’s NGS basics page splits the same process into four, adding nucleic acid isolation at the front. The stages below follow that four-step split.
- Nucleic acid isolation and purification. DNA is extracted from the sample and purified.
- Library preparation. Thermo Fisher lists fragmentation, adapter ligation, size selection, quantification and pooling as the elements of this stage. Pooling is what lets many samples share one run.
- Sequencing. The pooled library is clonally amplified, read by sequencing by synthesis, and converted from signal to bases (base calling). The instrument writes reads with quality scores to FASTQ files. Chemistry for one platform family is covered in Illumina (Solexa) sequencing.
- Data analysis. Reads are demultiplexed back to samples, filtered on quality, aligned to a reference (producing SAM/BAM files) and, where the project calls for it, variant-called (producing VCF files).
Output and how to read it
Each stage of the analysis leaves a file with a different job. Reading them in order tells you where a problem entered.
| Stage | File | What to check | CASRAI guide |
|---|---|---|---|
| Sequencing / base calling | FASTQ | Per-base quality scores before anything else | FASTQ format explained, Phred quality scores |
| Alignment | SAM / BAM | How reads map to the reference; choose a container format | SAM vs BAM vs CRAM |
| Variant calling | VCF | Whether there is enough coverage to trust a call at that position | Depth vs breadth of coverage |
The linked CASRAI guides explain how to interpret each file.
Troubleshooting
Reads assigned to the wrong sample
Because libraries from many samples are pooled and read together, an apparent sample mix-up can come from the multiplexing step rather than the biology. Index hopping in NGS explains how to detect it.
Weak calls at a position of interest
Check depth and breadth of coverage at that position before interpreting the variant file; see depth vs breadth of coverage.
Low-quality bases
Quality filtering is its own step in the analysis stage. Read the Phred scores in the FASTQ file first, using the guide linked above.
Free alternatives to generating new data
| Option | What the source says | Source |
|---|---|---|
| Reuse public reads from SRA | World’s largest publicly available repository of raw, unassembled sequence data | NCBI SRA documentation |
| Fetch the same data from a mirror | SRA is mirrored with EMBL-EBI (England) and NIG (Japan) via INSDC | NCBI SRA documentation |
| Controlled-access human data | Available only through dbGaP Authorized Access | NCBI SRA documentation |
| Single-target read with Sanger sequencing | Suited to a short, targeted read of one amplicon | Sanger sequencing guide |
FAQ
What is NGS DNA sequencing?
It is a massively parallel sequencing technology whose workflow consists of library preparation, sequencing and data analysis (Illumina).
Is “DNA sequencing NGS” a different method?
No. It is the same method under a reversed phrasing.
How much data does a run produce?
Illumina gives a range from 300 kilobases up to multiple terabases in a single run.
Where can I get existing NGS reads?
From the NCBI Sequence Read Archive and its INSDC mirrors; restricted human data goes through dbGaP Authorized Access.
How does it differ from Sanger sequencing?
NGS reads very large numbers of fragments in parallel instead of one at a time. Illumina notes that the Sanger-based Human Genome Project took over 10 years. See the DNA sequencing overview for the comparison of methods.
Sources (checked 2026-10-29)
- Illumina, Next-generation sequencing: https://www.illumina.com/science/technology/next-generation-sequencing.html
- NHGRI, DNA Sequencing Fact Sheet (updated June 27, 2023): https://www.genome.gov/about-genomics/fact-sheets/DNA-Sequencing-Fact-Sheet
- NCBI, Sequence Read Archive documentation: https://www.ncbi.nlm.nih.gov/sra/docs/
- Thermo Fisher Scientific, NGS basics (workflow steps only): https://www.thermofisher.com/us/en/home/life-science/sequencing/sequencing-learning-center/next-generation-sequencing-information/ngs-basics/what-is-next-generation-sequencing.html
