CommonNGS

Common problems in NGS data generation and analysis

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Contamination

• At least 7% of 1000 Genomes Project samples were contaminated with Mycoplasma.
• About 11% of NCBI SRA rodent and primate RNA-Seq samples were contaminated with Mycoplasma.
• Contamination detection should be performed before library construction to avoid a waste of time and money.
• Systematic contamination detection pipeline should also be performed once the data come back.

Sequencing depth

• Different library types require different sequencing depth. New sequencing methods should be evaluated by saturation curve.
• Sequencing depth also depends on the purpose.
  • ENCODE recommends that each replicate of WGBS should have 30x coverage;
  • For DMR identification, sequencing at levels higher than 5-15x leads to wasted resources that would be better spent on an increased number of biological replicates.
  • If the goal is primarily to identify long DMRs with large methylation differences, 1-2x per sample is acceptable.

Quality control of enrichment

• Lorenz curve can be used to evaluate the enrichment strength.
• For DNA-based enrichment, deeptools can be used.
• For RNA-based enrichment, I have written a simple script to normalize different expression levels using the paired Input sample. Please refer to MENG for more information.

Different genome and annotation versions

• There are different reference assemblies for the same species. Be sure to use the currect one. Coordinates from different genome assemblies can be transformed using LiftOver or CrossMap.
• There are also different sources of gene annotation, with different sensitivity and specificity.

Statistics

• Simple fold change will lead to enrichment of lowly expressed genes because of their larger variances. Be sure to use statistics such as negative binomial distribution in DESeq2 to find the differences.

Excel text format

• Using Excel to store the final results with default format will affect at least 30 gene names which will be converted to date format and are irreversible.

OS dependent line breaks

• Dos uses \r\n for line breaks, while Linux uses ‘\n’ for line breaks, which can lead to mistakes when files are transferred between different OS.
• dos2unix and unix2dos can be used easily to solve the problem.