title: COI sequence of insectivorous bird: fecal sample
design: We used a forward primer with the sequence: ACACTGACGACATGGTTCTACA GGTCAACAAATCATAAAGATATTGG (linker portion is underlined) and a reverse primer with the sequence TACGGTAGCAGAGACTTGGTCTGGWACTAATCAATTTCCAAATCC (Jusino  2019). First stage PCR amplifications were performed in 10 microliter reactions in 96-well plates, using MyTaq HS 2X mastermix (Bioline). PCR conditions were 5 cycles of: 95C for 60 s, 45C for 90 s, 72C for 90 s; 28 cycles of: 94C for 60 s, 50C for 90 s, 72C for 60 s. Subsequently, a second PCR amplification was performed in 10 microliter reactions in 96-well plates. A mastermix for the entire plate was made with MyTaq HS 2X mastermix. Each well received a separate primer pair with a unique 10-base barcode, which was obtained from the Access Array Barcode Library for Illumina (Fluidigm, South San Francisco, CA; Item# 100-4876). These primers contained the CS1 and CS2 linkers at the 3 ends of the oligonucleotides. Cycling conditions were: 95C for 5 minutes, followed by 8 cycles of 95C for 30, 60C for 30 and 72C for 30. Samples were subsequently pooled in equal volume using an EpMotion5075 liquid handling robot (Eppendorf, Hamburg, Germany). The pooled library was purified to remove fragments smaller than 200 bp using an AMPure XP cleanup protocol (1X, vol/vol; Agencourt, Beckmann-Coulter). The pooled libraries, with a 20% phiX spike-in, was loaded onto an Illumina MiniSeq mid-output flow cell (2x153 paired-end reads). The amplicons (before purification) were-pooled based on the distribution of reads per barcode, to generate a more balanced distribution of reads. The re-pooled library was purified using AMPure XP cleanup, as described above. The re-pooled libraries, with a 20% phiX spike-in, were loaded onto a MiniSeq flow cell, and sequenced (2x153 paired-end reads). Fluidigm sequencing primers (targeting the CS1 and CS2 linker regions) were used to initiate sequencing. De-multiplexing of reads was performed on instrument