BOL: Related items

Trust But Verify: Sequencing Your Cell Lines Might Reveal an Uninvited Guest

LEGE — Wed, 04 Jun 2025 00:07:57 -0500

High-throughput sequencing has become indispensable in cell biology, enabling detailed insights into chromatin structure, gene expression, and regulatory dynamics. Yet, when faced with unexpectedly low mapping rates to the human genome, researchers often rush to troubleshoot technical parameters—sequencer quality, adapter trimming, or aligner settings.

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.

Sequencing Solutions to World Health

Rahul Agarwal — Thu, 29 Aug 2013 15:05:35 -0500

"New technology that quickly, easily and economically reveals the genomes of viruses and pathogens transforms public health and medicine."

Source: Life technologies

Address of the bookmark: http://www.lifetechnologies.com/global/en/home/communities-social/blog/blogs/sequencing-solutions-to-world-health.html?cid=social_blogseries_20130829_11098264

Genome Browsers

Rahul Agarwal — Fri, 16 Aug 2013 19:04:47 -0500

Genome Browser is the platform/database used for searching and retreiving sequences and annotation of genomes belong to various eukaryotes, prokaryotes, etc.

Following are the weblink for different available browsers:

http://www.ensembl.org/index.html

http://ensemblgenomes.org/

http://genome.ucsc.edu/

http://www.ncbi.nlm.nih.gov/genome

http://www.ebi.ac.uk/genomes/

http://flybase.org/

http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi

http://www.sanger.ac.uk/resources/databases/

RNA Bioinformatics and High Throughput Analysis Jena

Sat, 09 Nov 2013 20:03:56 -0600

Research Topics:

High Throughput Sequencing Analysis
Comparative Genomics
Identification and Annotation of Non-coding RNAs
Bioinformatic Analysis and System Biology of Viruses
Coevolution of Proteins and RNAs
Algorithmic Bioinformatics
Phylogenetic Analysis

http://www.rna.uni-jena.de/index.php

Bio-Rad Acquires GnuBIO

Rahul Agarwal — Sat, 19 Apr 2014 10:36:36 -0500

http://www.businesswire.com/news/home/20140411005331/en/Bio-Rad-Acquires-GnuBIO-Developer-Droplet-Based-DNA-Sequencing#.U1KXnPm1b8o

Deadly Human Pathogen Cryptococcus Sequenced

Rahul Agarwal — Fri, 25 Apr 2014 11:02:21 -0500

"Now, researchers have sequenced the entire genome and all the RNA products of the most important pathogenic lineage of Cryptococcus neoformans, a strain called H99. The results, which appear in PLOS Genetics, also describe a number of genetic changes that can occur after laboratory handling of H99 that make it more susceptible to stress, hamper its ability to sexually reproduce and render it less virulent."

Source:

http://www.biosciencetechnology.com/news/2014/04/deadly-human-pathogen-cryptococcus-fully-sequenced

Paper:

http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1004292

Drawback of Exome Sequencing

Rahul Agarwal — Mon, 02 Jun 2014 05:46:43 -0500

Dr Eric Londin, Assistant Professor, Thomas Jefferson University, USA, stated that analysis of 44 exome datasets from four different testing kits showed that they missed a high proportion of clinically relevant regions in the 56 ACMG genes. "At least one gene in each exome method was missing more than 40 percent of disease-causing genetic variants, and we found that the worst-performing method missed more than 90 percent of such variants in four of the 56 genes," he says.

Source: http://www.eurekalert.org/pub_releases/2014-05/esoh-pco052914.php

GraphMap - A highly sensitive and accurate mapper for long, error-prone reads

Rahul Agarwal — Mon, 06 Jul 2015 08:46:53 -0500

GraphMap is a novel mapper targeted at aligning long, error-prone third-generation sequencing data.
It is designed to handle Oxford Nanopore MinION 1d and 2d reads with very high sensitivity and accuracy, and also presents a significant improvement over the state-of-the-art for PacBio read mappers.

GraphMap was also designed for ease-of-use: the default parameters can handle a wide range of read lengths and error profiles, including: Illumina, PacBio and Oxford Nanopore.
This is an especially important feature for technologies where the error rates and error profiles can vary widely across, or even within, sequencing runs.

http://biorxiv.org/content/early/2015/06/10/020719

Address of the bookmark: https://github.com/isovic/graphmap

GRASS: a generic algorithm for scaffolding next-generation sequencing assemblies.

Abhimanyu Singh — Tue, 23 May 2017 05:20:32 -0500

GRASS (GeneRic ASsembly Scaffolder)-a novel algorithm for scaffolding second-generation sequencing assemblies capable of using diverse information sources. GRASS offers a mixed-integer programming formulation of the contig scaffolding problem, which combines contig order, distance and orientation in a single optimization objective. The resulting optimization problem is solved using an expectation-maximization procedure and an unconstrained binary quadratic programming approximation of the original problem. We compared GRASS with existing HTS scaffolders using Illumina paired reads of three bacterial genomes. Our algorithm constructs a comparable number of scaffolds, but makes fewer errors. This result is further improved when additional data, in the form of related genome sequences, are used.

Address of the bookmark: https://github.com/AlexeyG/GRASS

LRSDAY: Long-read Sequencing Data Analysis for Yeasts

Poonam Mahapatra — Mon, 26 Aug 2019 18:07:33 -0500

Long-read sequencing technologies have become increasingly popular in genome projects due to their strengths in resolving complex genomic regions. As a leading model organism with small genome size and great biotechnological importance, the budding yeast, Saccharomyces cerevisiae, has many isolates currently being sequenced with long reads.

Address of the bookmark: https://github.com/yjx1217/LRSDAY