MGI stool metagenome sequecing

The required service is extension to metagenome sequencing that took place in 2021 by MGI Tech Latvia on >5000 gut microbiota samples from the Health and Early Life (HELMI) birth cohort NCT03996304. In total, we have collected approx. 10,000 stool samples from infants and their parents in the HELMI cohort. Now we want to extend the previously sequenced dataset by sequencing additional, earlier timepoints (3 and 6 week old infant stool samples) from the same study subjects. Since these new samples are from the same subjects, it is necessary to use the same, i.e. MGI, sequencing technology for the sake of data comparability. It is not possible to obtain comparable sequencing data with other sequencing technologies. The required service consists of shotgun metagenome sequencing of stool DNA samples extracted from 3 and 6 week old infants. Samples are already pipetted to plates according to MGI metagenome sequencing guidelines; we have in total 1969 samples in 21 plates that each have empty wells on positions 2D and 8D (42 in total), and in addition 5 bridging samples from the 2021 sequenced bacth, summing up to 1974 samples to pay. The samples to be sequenced are collected from newborns and they have for biological reason low DNA content and additionally have somewhat variable qualities as specified below. Hence, we want to apply a customised two stage pricing, which consists of 1) library prep and quality control for all samples, and 2) sequencing of libraries that pass the QC. Because the number of samples passing to stage 2 can be only estimated, the total value of the procurement is also an estimate.

The required service consists of shotgun metagenome sequencing of stool DNA samples extracted from 3 and 6 week old infants. Samples are already pipetted to plates according to MGI metagenome sequencing guidelines; we have in total 1969 samples in 21 plates that each have empty wells on positions 2D and 8D (42 in total), and in addition 5 bridging samples from the 2021 sequenced bacth, summing up to 1974 samples to pay. As the DNA yield in the newly colonized infants is low and hence majority of samples do not meet the minimum DNA content ≥0.5μg to reach category “qualified” of the MGI sample submission guidelines, the service must be broken to two steps as follows: 1) library prep and quality control for all samples, and 2) sequencing of libraries that pass the QC. In the previous MMHP sequencing round in Riga in 2020-2021 with >4000 infant samples sequenced from the same cohort, there was a lack of correlation between the DNA concentration and QC category/success of sequencing. This might partly be due to higher host DNA in infant samples when the microbiota has just started to develop. Also, due to the simplicity of the community, less than 6GB will allow decent characterisation of the microbiota in samples that cannot reach that depth. For these and below specified technical factors related to the accuracy of the DNA concentrations, we see it valuable to attempt to make a library of all samples independent of their DNA concentration. Conditions in which the samples will be sent for service: We have measured the DNA content of the samples with fluorometric methods, but it is not reliable for two reasons: 1) the consentration measurent of samples with low DNA concentrations is not reliable due to methdological limitations 2) The DNA preps have been stored in -20C since 2018-2020, and despite precautions, some evaporation had happened. Hence, some samples are more concentrated than originally measured. The samples were prepared so that samples exceeding 27 ng/ul were diluted with water to 27 ng/ul and 34-35 ul pipetted to each well, and the ones with lower concentrations or volume were taken as is, with miminum volume of 5 ul. Out of the 1969 infant samples, 1352 have < 500 ng DNA, and 152 samples <10 ng based on the original measurements. Anonymised sample list with measured concentrations and dilution conditions are attached. Similar to the variable DNA content, the EDTA content of the preps is variable as depending on the DNA content of the samples as the samples were treated the sample in one of these 3 ways. All samples were originally eluted in the elution buffer (EB) containing 10mM Tris-HCl, 0,5 mM EDTA (pH 8): 1) Samples in EB, diluted in water 2) Samples that were too dilute to be further diluted, hence in 0.5 mM EDTA. 3) Some wells on the elution plates had evaporated, and we reconstituted the samples by adding water. Hence, they have an unknown and variable amount of EDTA. We do not recommend buffer exchange as quite many samples are already on the lower limits of concentration/DNA left and in our experience there is always a loss of DNA in buffer enhance, even with beads. Since summer 2023 the samples are ready for shipment in 96-plates compatible with MGI pipeline. The samples will be shipped as is to prevent well-to-well prevent contamination and we cannot make their quantity and/or quality closer to the requirents.