Evaluation of methods for the reduction of contaminating host reads when performing shotgun metagenomic sequencing of the milk microbiome

peer reviewedShotgun metagenomic sequencing is a valuable tool for the taxonomic and functional profiling of microbial communities. However, this approach is challenging in samples, such as milk, where a low microbial abundance, combined with high levels of host DNA, result in inefficient and uneconomical sequencing. Here we evaluate approaches to deplete host DNA or enrich microbial DNA prior to sequencing using three commercially available kits. We compared the percentage of microbial reads obtained from each kit after shotgun metagenomic sequencing. Using bovine and human milk samples, we determined that host depletion with the MolYsis complete5 kit significantly improved microbial sequencing depth compared to other approaches tested. Importantly, no biases were introduced. Additionally, the increased microbial sequencing depth allowed for further characterization of the microbiome through the generation of metagenome-assembled genomes (MAGs). Furthermore, with the use of a mock community, we compared three common classifiers and determined that Kraken2 was the optimal classifier for these samples. This evaluation shows that microbiome analysis can be performed on both bovine and human milk samples at a much greater resolution without the need for more expensive deep-sequencing approaches.Irish Dairy Lev

Maternal and fetal blood lipid concentrations during pregnancy differ by maternal body mass index: findings from the ROLO study

Abstract Background Pregnancy is a time of altered metabolic functioning and maternal blood lipid profiles change to accommodate the developing fetus. While these changes are physiologically necessary, blood lipids concentrations have been associated with adverse pregnancy outcomes such as gestational diabetes, pregnancy-induced hypertension and high birth weight. As blood lipids are not routinely measured during pregnancy, there is limited information on what is considered normal during pregnancy and in fetal blood. Methods Data from 327 mother-child pairs from the ROLO longitudinal birth cohort study were analysed. Fasting total cholesterol and triglycerides were measured in early and late pregnancy and fetal cord blood. Intervals were calculated using the 2.5th, 50th and 97.5th centile. Data was stratified based on maternal body mass index (BMI) measured during early pregnancy. Differences in blood lipids between BMI categories were explored using ANOVA and infant outcomes of macrosomia and large-for-gestational-age (LGA) were explored using independent student T-tests and binary logistic regression. Results All maternal blood lipid concentrations increased significantly from early to late pregnancy. In early pregnancy, women with a BMI  30 kg/m2) had higher concentrations than both women in the normal-weight and overweight category in early and late pregnancy (P < 0.001 and P = 0.03, respectively). In late pregnancy, triglyceride concentrations remained elevated in women in the obese category compared to women in the normal-weight category (P = 0.01). Triglyceride concentrations were also elevated in late pregnancy in mothers that then gave birth to infants with macrosomia and LGA (P = 0.01 and P = 0.03, respectively). Conclusion Blood lipid concentrations increase during pregnancy and differ by maternal BMI. These intervals could help to inform the development of references for blood lipid concentrations during pregnancy. Trial registration ROLO Study - ISRCTN54392969 . Date of registration: 22/04/2009

Scatter plot displaying the association between birth weight and late pregnancy triglyceride concentrations stratified by BMI category.

<p>Scatter plot displaying the association between birth weight and late pregnancy triglyceride concentrations stratified by BMI category.</p

Multiple regression model examining the associations between maternal blood lipids and 6-month infant weight as an outcome.

<p>Multiple regression model examining the associations between maternal blood lipids and 6-month infant weight as an outcome.</p

Scatter plot displaying the association between birth weight and cord triglyceride concentrations stratified by BMI category.

<p>Scatter plot displaying the association between birth weight and cord triglyceride concentrations stratified by BMI category.</p

Characteristics of mothers and infants.

<p>Characteristics of mothers and infants.</p

Multiple regression model examining the associations between maternal blood lipids and birth weight as an outcome.

<p>Multiple regression model examining the associations between maternal blood lipids and birth weight as an outcome.</p

Concentrations of blood lipids (Median (IQR).

<p>Concentrations of blood lipids (Median (IQR).</p