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: http://bioinformatics.oxfordjournals.org/content/early/2014/02/12/bioinformatics.btu078.full.pdf

Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes

Mads Albertsen, Philip Hugenholtz, Adam Skarshewski, Gene W. Tyson, Kåre L. Nielsen and Per .H. Nielsen

Nature Biotechnology 2013, doi: 10.1038/nbt.2579

Address of the bookmark: https://github.com/MadsAlbertsen/multi-metagenome

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.

Handle sequence locations for bioinformatics http://www.ingolia-lab.org/seqloc-tutorial.html

Address of the bookmark: http://www.stackage.org/snapshot/nightly-2014-12-28/package/seqloc-0.6

Tutorial at http://evomics.org/learning/genomics/satsuma/

Address of the bookmark: http://satsuma.sourceforge.net/

Krona

https://sourceforge.net/p/krona/home/krona/

Address of the bookmark: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053813/

FSA brings the high accuracies previously available only for small-scale analyses of proteins or RNAs to large-scale problems such as aligning thousands of sequences or megabase-long sequences. FSA introduces several novel methods for constructing better alignments:

• FSA uses machine-learning techniques to estimate gap and substitution parameters on the fly for each set of input sequences. This "query-specific learning" alignment method makes FSA very robust: it can produce superior alignments of sets of homologous sequences which are subject to very different evolutionary constraints.
• FSA is capable of aligning hundreds or even thousands of sequences using a randomized inference algorithm to reduce the computational cost of multiple alignment. This randomized inference can be over ten times faster than a direct approach with little loss of accuracy.
• FSA can quickly align very long sequences using the "anchor annealing" technique for resolving anchors and projecting them with transitive anchoring. It then stitches together the alignment between the anchors using the methods described above.
• The included GUI, MAD (Multiple Alignment Display), can display the intermediate alignments produced by FSA, where each character is colored according to the probability that it is correctly aligned (see the picture and movie at the top of the page).

You can see more information on the FAQ. 

Address of the bookmark: http://fsa.sourceforge.net/

Address of the bookmark: http://www.isical.ac.in/~bioinfo_miu/FOGSAA.htm