Control males had a significantly lower calling rate than fed males (Fed1 and Fed2 pooled; paired t-test: N = 8, T = -3.1, p < 0.05; Fig. 3).
Paired-end libraries were prepared by using the Nextera DNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA) and sequenced in two lanes on an Illumina HiSeq2000 at the 12-plex level of multiplexing, producing from 30 to 50 million reads per pool (paired-end 100-nt set-up).
Ten independent hybridizations were carried out, and the expression levels of all genes were determined for each "sample/control" pair and for pool pairs.
One of the breast cancer pool pairs gave log-ratios that were comparatively highly variable, and is excluded from all analyses.
Hence, we contrasted pools formed by equal plasma volumes from 100 cases or from 100 pair-matched controls, with eight such pool pairs for each of the study diseases.
We used 3,200 patient samples (800 stroke cases, 800 CHD cases, and 1,600 controls) to form case and control pool pairs for 16 IPAS experiments (8 stroke + 8 CHD).
Following application of an additional requirement that proteins were quantified for at least two of the pool pairs for a disease, 346 proteins for CHD and 366 proteins for stroke were included in statistical analyses.
Since MHC blood type (BT) is known to affect MD resistance, a total of 18 independent pool pairs were constructed according to family × BT combination, with some combinations represented twice for technical reasons.
In a first stage, five pairs of total RNA samples were used for whole-transcriptome analysis: four pairs that each consisted of the RNA from a patient with PD and the RNA from a healthy volunteer with matching age and sex and one pool pair that consisted of a pool of the total RNA from all patients investigated and a pool of the total RNA from the healthy controls.
Six other proteins have a FDR < 0.20 for CHD association, and 14 have a FDR < 0.20 for stroke association. Figure 1 shows peptide coverage and case versus control concentration ratios for B2M, ORM1, PPIA, and IGFBP4 separately for each plasma pool pair.
