Name		Snorre Flo
Affiliations	The University Centre in Svalbard (UNIS), UiT The Arctic University of Norway (UiT), The Nansen Legacy
Email		snorref@unis.no, snorreflo@gmail.com
Date		13.07.2023
Dataset		copdiet_novaseq_raw
Files		copdiet_novaseq_readme.txt, MD5.txt, ngsfilter_mappings (copdiet_novaseq_all_ngsfilter.xls, SNO1_ngsfilter.txt, SNO2_ngsfilter.txt, SNO3_ngsfilter.txt, SNO4_ngsfilter.txt, SNO5P1_ngsfilter.txt and SNO5P2_ngsfilter.txt), metadata (copdiet_novaseq_raw_meta.xls, copdiet_novaseq_raw_meta.csv), zipped data libraries (SNO2 + SNO5P2: X204SC22031183-Z01-F001_01.zip, SNO3: X204SC22031183-Z01-F001_02.zip, SNO1: X204SC22031183-Z01-F001_03.zip, SNO5P1: X204SC22031183-Z01-F001_04.zip, SNO4: X204SC22031183-Z01-F001_05.zip)
Description	The copdiet_novaseq_raw dataset contains DNA reads from a total of 461 samples including extraction negatives, PCR negatives and copepods. The purpose of the study was to investigate the trophic interactions of three small Arctic copepod species: Oithona similis, Microsetella norvegica, Microcalanus spp. (M. pusillus and/or M. pygmaeus) using a "brute force" prey metabarcoding approach. Zooplankton were collected on three locations (process stations P1, P4 and P7) along a transect from the Barents Sea to the Arctic Nansen Basin in August 2019 (Q3), December 2019 (Q4), March 2021 (Q1) and April/May 2021 (Q2) with 64 um Bongo-nets vertical hauls from 1000m/sea-floor to surface, and fixed on ice-cold ethanol (96%, -20 deg C). Up to 14 biological replicates were picked of each species from each station from each cruise, and the samples are named accordingly (i.e. os_1_7_13 contains Oithona similis from cruise Q1, station P7, and biological replicate number 13). Other sample name identifiers include mn (M. norvegica), mp (Microcalanus spp.), en (extraction negative) and pneg (PCR plate negative). DNA from picked specimen were extracted using the E.Z.N.A Tissue DNA kit (Omega Bio-Tek), and tested for contamination by amplification of V7 18S SSU rDNA and subsequent 1% gel-electrophoresis. We amplified the 18S SSU rRNA V7 region (~100 – 110 bp) with 18S_allshorts primers (Forward 5’-TTTGTCTGSTTAATTSCG-3’, and Reverse 5’-GCAATAACAGGTCTGTG-3’, Guardiola et al., 2015) and sequenced cleaned product on two lanes of the Illumina Novaseq platform using 150 PE chemistry. The raw data was explored and processed on an HPC into a zOTU-table with sample-wise numerical abundances and taxonomy from the Protist Ribosomal database (PR2/v.4.14.0), using a combination of FastQC/0.11.9, BLAST+/2.8.1, OBITools/1.2.12 and VSEARCH/2.9.1 utilities. More information about processing, including scripts used - is openly available on github (https://github.com/snflo/bruteforce). 
User guide	Accompanying files include the lib_ngsfilter.txt mapping-files (with sample ids, primer-tags, forward and reverse primer sequences) used to assign reads to samples. Its important to pair the ngsfilter sample mapping files (i.e. SNO1_ngsfilter.txt) with the matching forward (SNO1_EKDL220002996-1a-4_HHMCJDSX3_L1_1.fq) and reverse (SNO1_EKDL220002996-1a-4_HHMCJDSX3_L1_2.fq) library partitions, as the samples were sequenced using the same set of tagged primers. Failing to match the correct mapping file with its library will lead to reads being assigned to the wrong samples. Other tools may achieve the same goal, but we used the illuminapairedend function (OBITools) to pair forward and reverse reads, and the ngsfilter command (OBITools) to assign reads to their respective samples.