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Introduction to Sequencing Platform

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Do you need biological repetition and how many are suitable?

For data quality, results output and successful submission, we must pay attention to the problem of biological duplication. Taking into account individual differences, the late inter-group statistical analysis and deviation samples, the natural environment at least 3 biological replicates, recommend 5; if the human intestine, feces and other samples, due to the high individual specificity, it is recommended to repeat more than 10. From a scientific point of view, it is best to sequence and analyze the whole batch of samples at the same time, which can not only reduce the systematic error between different batches, but also save the project cycle. If the sample preparation is difficult, it can also be started in batches, but this may cause system error problems.

If we are concerned about the diversity of eukaryotic microorganisms in the environment, which is better, 18S sequencing or ITS sequencing?

ITS sequencing is mainly aimed at the study of fungal diversity, with high annotation accuracy; 18S sequencing is aimed at eukaryotic microorganisms, with a wide range of annotations, but the accuracy is relatively low for fungi; the sequencing scheme can be reasonably selected according to the research purpose.

What is the difference between amplicon sequencing and metagenome sequencing?

Amplicons are mainly divided into 16S, 18S, ITS and other sequencing, focusing on the composition of species in environmental samples, the technology is inexpensive and practical. Metagenomics focuses on the information of species composition, functional composition and metabolic pathways in environmental samples, and this technology digs deep into the functional information of environmental samples.

What is the difference between DGGE technology and high-throughput sequencing technology in the study of environmental microbial community diversity?

Molecular fingerprinting techniques such as DGGE often contain only dozens of bands in their experimental results, which can only reflect the dominant population in the sample, require standard strains, and are limited by the characteristics of gel electrophoresis, unable to detect the types of rare flora, so its repeatability and resolution are not ideal. The high-throughput sequencing technology can simultaneously detect the dominant population and trace bacteria in the sample, obtain the composition of the microbial community in the sample, and digitize its content.

What is the sequencing length that amplicon sequencing can satisfy?

The sequencing length of Miseq PE300 platform is 600bp, but its sequencing quality is poor, and quality control needs to filter out sequences with poor quality over 100bp. Hiseq2500 PE250 platform has good sequencing quality, the total sequencing length is 500bp, leaving only about 460bp after removing 12bp barcode and some sequences with poor quality. Therefore, for the sake of conservation, the length of the amplicon fragment should not exceed 460bp, otherwise the double-end sequencing results will not be spliced, and attention should be paid to the selection of primers.

In 2013, Henke et al. summarized the advantages of genome-wide BSA over transcriptome (RNAseq) for trait mapping.

1. Coverage: Whole genome BSA can cover the entire genome, while RNAseq only sequences the coding region. 2. The preference of gene detection: the whole genome BSA is more uniform, no preference, and is not affected by time or organization. RNAseq is biased towards genes that are highly expressed at a particular time or tissue. 3. Regional preference: gene distribution is uneven. If the gene density in some regions is low, RNAseq may be missed. 4. Polymorphism: Most of the polymorphisms in the genome are located in the non-coding region, and the polymorphism detected by RNAseq is low, and only a few polymorphic markers linked to the mutation site can be detected by RNAseq. Therefore, it is easier to detect polymorphic markers linked to target genes using genome-wide BSA.

Can artificial mutagenesis only be EMS mutagenesis? Can BSA trait be used to locate traits obtained by other mutagenesis methods?

BSA trait mapping is based on SNP, which is analyzed by comparing SNP frequency differences. EMS induces point mutations, so EMS-induced mutations are suitable for this analysis method. Most of the other mutagenesis methods induce structural variation, and the region that leads to the difference of target traits may not be found at the SNP level, so it is not suitable for SNP frequency analysis.

Can BSA trait mapping be performed in polyploid species?

Already assembled polyploid reference genome species, e.g., "oilseed rape, tobacco, cotton", can be used for trait mapping with BSA.

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