Dneasy power kit

DNA was extracted from swabs using the Blood and Tissue Kit (Qiagen). Briefly, cotton tips were incubated for 1 h at 56 °C in 400 µl Lysis buffer with 60 µl of proteinase K (20 mg/ml). After a 10-minute centrifugation step at 7000 g, the resulting solution was processed according to the manufacturer’s protocol. For DNA extraction from water, samples were first defrosted and filtered through Millipore Stericup filters (0.22 µm). The filters were cut into small fragments and DNA was extracted with the DNEasy Power Kit (Qiagen) according to the manufacturer’s protocol.

The fifth instar eri silkworm larvae were used for metagenomics analysis of bacteria and five healthy larvae of approximately the same size were used. Insect dissection was carried out as described earlier. Total genomic DNA to be used for metagenomics analysis was pooled by direct extraction of DNA from the midgut and hindgut (to cutter for both cultivable and uncultivable bacteria) and from 24 h nutrient broth-enriched growth using homogenates from the respective gut compartments (to enrich cultivable bacteria) using a DNeasy Power Kit (Qiagen, USA). The DNA was later pooled and quantified using a Qubit Fluorimeter (V.3.0). TheV3-V4 region of 16S rRNA was amplified using specific V3 Forward primer- (5′-CCTACGGGNBGCASCAG) and V4 Reverse primer- (5′-GACTACNVGGGTATCTAATCC). The amplified product was checked on 2% agarose gel and gel purification was done to remove non-specific amplifications. In addition, 5 ng of amplified product was used for library preparation using the NEBNext Ultra DNA Library Preparation Kit (New England Biolabs Inc., USA). The library quantification and quality estimation were done in Agilent 2200 TapeStation. The prepared library was sequenced in Illumina HiSeq 2500 with 2*250 cycles chemistry.