More at http://catchenlab.life.illinois.edu/stacks/

Address of the bookmark: http://catchenlab.life.illinois.edu/stacks/

Cleaning your data in this way is often required: Reads from small-RNA sequencing contain the 3’ sequencing adapter because the read is longer than the molecule that is sequenced. Amplicon reads start with a primer sequence. Poly-A tails are useful for pulling out RNA from your sample, but often you don’t want them to be in your reads.

Cutadapt helps with these trimming tasks by finding the adapter or primer sequences in an error-tolerant way. It can also modify and filter reads in various ways. Adapter sequences can contain IUPAC wildcard characters. Also, paired-end reads and even colorspace data is supported. If you want, you can also just demultiplex your input data, without removing adapter sequences at all.

Cutadapt comes with an extensive suite of automated tests and is available under the terms of the MIT license.

If you use cutadapt, please cite DOI:10.14806/ej.17.1.200 .

Address of the bookmark: https://cutadapt.readthedocs.io/en/stable/installation.html#quickstart

Wgsim is a small tool for simulating sequence reads from a reference genome. It is able to simulate diploid genomes with SNPs and insertion/deletion (INDEL) polymorphisms, and simulate reads with uniform substitution sequencing errors. It does not generate INDEL sequencing errors, but this can be partly compensated by simulating INDEL polymorphisms.

Wgsim outputs the simulated polymorphisms, and writes the true read coordinates as well as the number of polymorphisms and sequencing errors in read names. One can evaluate the accuracy of a mapper or a SNP caller with wgsim_eval.pl that comes with the package.

Address of the bookmark: https://github.com/lh3/wgsim

Address of the bookmark: https://github.com/marbl/Krona/wiki

Address of the bookmark: http://pacb.com/blog/the-hifi-difference-true-long-reads-vs-synthetic-long-reads/

Before you go down that path, consider this critical biological question:
Are you sequencing human cells—or bacterial contamination?

The Silent Saboteur: Mycoplasma in Cell Cultures

Mycoplasma contamination remains one of the most widespread and underdiagnosed issues in tissue culture work. Studies suggest that 15–35% of cell lines in use may be contaminated, often without visible signs. Unlike other microbial infections, Mycoplasma does not produce cloudiness, odor, or a change in pH. Many researchers won’t detect it unless they specifically test for it.

The consequences, however, are profound. Mycoplasma can significantly alter:

• Host gene expression patterns

• Cell proliferation rates

• Epigenetic profiles and chromatin accessibility

• Cytokine signaling and immune responses

In short, it can skew your results, compromise your biological conclusions, and invalidate weeks or months of research.

A Simple Diagnostic Step: Map Against Mycoplasma Genomes

If you encounter poor alignment rates to the human genome, consider mapping your reads to a Mycoplasma reference genome—or better yet, use a combined human + Mycoplasma reference. There have been cases where over half of all reads, initially assumed to be from human cells, were in fact bacterial in origin. This check is fast, easy, and could save your project.

How Contamination Happens—and Persists

Mycoplasma is small (0.1–0.3 μm), lacks a cell wall, and can pass through standard filters undetected. Common sources include:

• Contaminated reagents (e.g., FBS)

• Infected cell lines obtained from other labs

• Poor aseptic technique or shared equipment

Once present, it spreads quickly between cultures and can persist for months, silently affecting results.

Why Treatment Is Difficult

While antibiotics such as Plasmocin or BM-Cyclin are sometimes used, they often offer only partial resolution and may themselves alter cell behavior. In many cases, the best course of action is to discard the contaminated culture and start with a fresh, verified stock.

Practical Recommendations for Researchers

• Routinely test for Mycoplasma using PCR, qPCR, or fluorescence-based assays

• Incorporate contamination screens into your sequencing QC pipeline

• Use combined reference genomes when mapping ambiguous reads

• Practice strict aseptic technique and monitor all incoming cell lines

• Don’t ignore unexplained data anomalies—they might point to contamination

Closing Thought: Contamination Is a Biological Variable

It’s easy to view poor mapping as a technical issue, but sometimes the problem lies deeper—in the biology itself. Mycoplasma contamination doesn’t just interfere with sequencing; it interferes with science. As a research community, we must treat contamination not as an afterthought, but as a key variable to control.

So next time your reads won’t align, don’t just tune the aligner. Ask if your cells are telling the truth—or if they're hiding something.

Address of the bookmark: https://github.com/rrwick/Unicycler

Address of the bookmark: http://hibberdlab.com/transrate/index.html

The pipeline is open-source and hosted in a public Bitbucket repository.

Address of the bookmark: https://tritexassembly.bitbucket.io/