FASTQExperiment Detail
| Title | R20160704-ST050_S50_L001 |
| Design Description | The filter samples were subjected to eDNA extraction following the method suggested by Yamanaka et al. (2017). The filter was rolled into acylindrical shape using sterile forceps and placed into a upper part of spin column (EZ-10; Bio Basic, Markham, Ontario, Canada) which was removed the silica membrane before use. After the any excess water remaining in the filters were removed by centrifuge for 1 min at 6000 g, the mixture of 200 uL of ultrapure water, 100 uL of Buffer AL, and 10 uL of proteinase K was dispensed onto the filter in each spin column and incubated for 15 min at 56 degC. The Buffer AL and proteinase K used in this step were supplied by the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). After the incubation, the spin columns were centrifuged for 1 min at 6000 g to elute the eDNA into 2-mL tube, a lower part of spin column. The upper part of spin column was then placed in a new 2-mL tube, and 200 uL of Tris-EDTA buffer (pH 8.0) was dispended onto the filter and incubated for 1min at room temperature to recover the remaining DNA on the filter. The spin columns were centrifuged for 1 min at 6000 g to obtain the second elution and was mixed with the first elution. Then, 100 uL of Buffer AL and 600 uL ethanol were added to each elutes in 2-mL tube and mixed well by pipetting. eDNA was collected and purified from each solution using DNeasy Blood and Tissue Kit following the manufacturer's protocol, with a minor modification in the final elution volume to 100 uL of Buffer AE. |
| Organism | lake water metagenome |
Library Description
| Name | R20160704-ST050_S50_L001 |
| Strategy | AMPLICON |
| Source | METAGENOMIC |
| Selection | PCR |
| Layout | PAIRED |
| Orientation | |
| Nominal Length | 0 |
| Nominal Sdev | |
| Construction Protocol | The first PCR was performed in 5 replicates in each of 12 uL reaction for a sample. The mixture of the reaction was as follows: 0.3 uM of PaaDlp-2_F, 0.15 uM of PaaDlp-2_R1 and R2 in 2x KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA), and 2 uL sample eDNA. To monitor cross-contamination during the library preparation, triplicated non template control (NTC) were included in the first PCR. The PCR thermal conditions were 3 min at 95 degC, 35 cycle of 20 s at 98 degC 15 s at 60 degC, and 15 s at 72 degC followed by the final extension for 5 min at 72 degC. The first PCR products were purified twice using the Agencourt AMPure XP (Beckman Coulter,USA) according to the manufacturer's instructions (target amplicon length; ca. 290 bp). The second PCR was performed in 3 replicates in each of 12 uL reaction for each replication of first PCR (total 15 replication). To distinguish samples for maltiplex sequencing, each one of the replicated first PCR product was indexed with different combinations of indexing primers. The mixture of the reaction was as follows: 0.3 uM of each second PCR primer in 2x KAPA HiFi HotStart ReadyMix, and 2 uL of purified first PCR product. As negative controls in the second PCR, 2 uL of the first PCR product of the NTC was added to each reaction instead of the real templates eDNA. The PCR thermal conditions were 3 min at 95 degC, 12 cycle of 20 s at 98 degC and 15 s at 65 degC, with the final extension for 5 min at 72 degC. |
Platform
| Platform | ILLUMINA |
| Instrument Model | Illumina MiSeq |
Processing
| Base Calls | |
|---|---|
| Sequence Space | |
| Base Caller | |
Spot Information
| Number of Reads per Spots | 0 |
| Spot Length | 302 |
Read Spec
| Read Index 0 | |
|---|---|
| Read Label | |
| Read Class | Application Read |
| Read Type | Forward |
| Base Coord | 1 |
| Read Index 1 | |
| Read Label | |
| Read Class | Application Read |
| Read Type | Reverse |
| Base Coord | 152 |
