Technical support/Common problem


Microbial diversity

Question 1:What is Reads, Index, Barcode, Tag, OTU?

answer:Reads: the sequence label produced by high throughput sequencing platform is called reads; Barcode: synonymous with Index, refers to a small sequence of gene sequences used to distinguish different samples from the sequence of joint sequences during sequencing; Tag: a relatively short sequence, a pair of reads in PE sequencing, spliced through overlap between the two. A sequence, used for tagging other sequences; OTU (Operational Taxonomic Units): in phylogeny or population genetics research, in order to facilitate analysis, human artifice can be considered as the same marker for a species of taxa (strain, species, genus, grouping, etc.).

Question 2:What is the amount of data that is provided by the sequencing?

answer:Because of the different species composition and abundance of different environmental samples, the amount of sequencing needed is different. We will choose the appropriate sequencing depth to ensure that the number of sequencing items in a single sample is higher than 20 thousand tags, and the number of sequencing items can be increased according to customer requirements.

Question 3:How long is the cycle required for the project?

answer:When we receive the sample from the customer, the first time is to be tested. Under the condition of sample test, the project cycle is completed in 35 natural days normally.

Question 4:What are the sample requirements and standards?

answer:The concentration of DNA is more than 20ng/uL, the volume is more than 15uL, OD260/280=1.8~2.0, and DNA is not degraded. Samples of soil, feces and silt were more than 1G, and 2-5L samples were filtered. Samples were detected by Nanodrop detection system, agarose gel electrophoresis, Qubit, Caliper, Aglient 2100 and other detection methods.


Transcriptional regulation

Question 1:Are there any differences in the data of different samples during the sequencing process? If there are differences in the data of different samples, will it affect the result of comparative analysis?


During the sequencing process, there is a difference between the data produced by different samples, which is a normal phenomenon. This is because the difference in the amount of data between samples may make the mapped reads difference in each sample, but it does not necessarily affect the follow-up analysis such as gene quantitative analysis; for the transcriptional analysis, the quantitative results of the gene are more important and can be obtained from the satiety and curve through the quantitative results of the gene. When the quantitative results of each gene are saturated when each sample is in the condition of their own data, it is necessary to conduct accurate quantitative analysis.

Question 2:How to judge whether a gene is a differential gene? If it is a differential gene, how to determine whether the gene expression is up or down?


When judging whether a gene has a differential gene without biological repetition, it first uses TMM to standardize the readcount data, and then uses DEG-seq to analyze the difference with a screening threshold of Q-value <0. 005&|log2FoldChange|>1; for the differential gene, if the gene's log2 Foldchange>0, the difference gene is up up, and vice versa. If log2Foldchange <0 is considered, the differential gene is down regulated.

Question 3:How to evaluate whether RNA is qualified and meet the subsequent database test?


Because RNA is easy to degrade, we operate all the ice on the preparation of samples and transportation (using dry ice transport) and extraction. So before the library construction, we will strictly test the RNA samples:

• The degradation and pollution of RNA were judged by agarose gel electrophoresis. The criteria were 2 clear bands and 28S/16S 2:1 relationship.

• The purity of RNA was detected by NanoDrop, and the OD260/280 was more than 1.

• The integrity of RNA is accurately detected by Agilent 2100. The standard is RIN value greater than 6.5, the baseline of the map is unchanged, and the 5S peak is normal.