iSeq Test Sequence of Pool 2

Owned by Gregg Randolph
Last updated: Mar 22, 2021 by Alex Buerkle
1 min read

Pool Makeup Info

Pool 2 has all 8 MID plates in it. It is therefore the best confirmatory test.

Dilution and Prep for iSeq Sequencing

The aliquot of Pool2 size selected for the 300-450 bp range without dilution of initial pool qPCRs a concentration of 10.6 nM.

  1. Dilute non-diluted pool to 1 nM: Add 9.5 ul Pool 2 to 90.5 ul TE.

  2. Dilute to loading concentration: Add 10 ul from above to 90 ul 10uM Tris

  3. Equilibrate FlowCell to RT ~10 minutes

  4. Load 20ul onto iSeq cartridge

  5. Start Run

Bioinformatics

Raw and parsed data are in: /project/microbiome/data/seq/Alfalfa/GBS/Alf1GBS_iSeq

cut -f 1,2 -d ',' Alf1GbsTest_Demux.csv | sed -E 's/(.*)/prepend,\1/' > Alf1GbsTest_Demux_simplified.csv sbatch parse_barcodes_slurm.sh ## only took 86 minutes with the correct MID key # to monitor number of samples recovered before the parsereport is complete grep '@' parsed_AlfGbsTest_S1_L001_R1_001.fastq | sed -E 's/@(.*) --.*/\1/' | sort | uniq | wc -l

At least one read was recovered from every one of the 764 MIDs that were used and in the Demux key.

Note that we need to preprocess the Demux key (line 1 above), to get it into form that the parser expects (three columns, with second column being the MID, and the third column being the sample name). For example, see below.

index_10nt_10,CGTCAGCCAA,PEHA12_2_24
index_10nt_104,TCCTCTTGAA,PEAB2_1_08
index_10nt_11,TAGGAGCCAA,PEHA12_2_27

 

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