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Additional file 2. English editing certificate.

The sequence of primers for GBP-SubA and optimization of E. coli strain and vector of GBP-SubA expression. (DOC 710 kb)

The distribution of total reads alignments. The total reads of three samples with a 0.55 % expected proportion of H1N1 within mixed RNA samples were first aligned to reference genomes of human (UCSC hg19) by using Bowtie2 and BLASTn with default parameters. The unaligned reads were then aligned to a dataset including reference genomes of Mycoplasma (313 sequences, NCBI genome database), bacterial (3022 sequences, NCBI genome database), flu (246,715 sequences, EpiFlu), other viral (1,757,357 sequences, NCBI genome database), and the whole NCBI nucleotide (nt) database by using Bowtie2 with default parameters. The Mycoplasma reads ratios for three samples were No pretreatment: 1.39 %, BD: 1.80 %, BD + WTA(8 h): 0.14 %. In the category of ‘Other’, we found that most of assignment could be considered as artificial alignments (chicken, rabbit, fruit fly, vector, etc.) which might be attributed to sequence homology. Undefined: failed to be aligned. (DOCX 130 kb)

The missed and over-amplified nucleotide motifs with enrichment analysis. (DOCX 179 kb)

Ratios of mycoplasma-aligned reads in samples. (DOCX 16 kb)

Primers used for RT-PCR amplification of H1N1 genomic segments (DOCX 22 kb)

Single Nucleotide variations detected by next-generation sequencing and Sanger sequencing (DOCX 20 kb)

Single Nucleotide variations detected by next-generation sequencing and Sanger sequencing (DOCX 20 kb)

Serotyping by HA and NA segments. Expected proportions of H1N1 within mixed RNA samples of were 0.55Â % (a) and 1.5Â % (b). (DOCX 124 kb)

Primers used for RT-PCR amplification of H1N1 genomic segments (DOCX 22 kb)

Schematic diagram of experimental design and analysis. A549, human alveolar adenocarcinoma cell line. Pretreatments of background depletion (BD) and/or whole-transcriptome amplification (WTA) were applied to mixed samples before library preparation. (DOCX 147 kb)

The missed and over-amplified nucleotide motifs with enrichment analysis. (DOCX 179 kb)

Serotyping by HA and NA segments. Expected proportions of H1N1 within mixed RNA samples of were 0.55Â % (a) and 1.5Â % (b). (DOCX 124 kb)

The distribution of total reads alignments. The total reads of three samples with a 0.55 % expected proportion of H1N1 within mixed RNA samples were first aligned to reference genomes of human (UCSC hg19) by using Bowtie2 and BLASTn with default parameters. The unaligned reads were then aligned to a dataset including reference genomes of Mycoplasma (313 sequences, NCBI genome database), bacterial (3022 sequences, NCBI genome database), flu (246,715 sequences, EpiFlu), other viral (1,757,357 sequences, NCBI genome database), and the whole NCBI nucleotide (nt) database by using Bowtie2 with default parameters. The Mycoplasma reads ratios for three samples were No pretreatment: 1.39 %, BD: 1.80 %, BD + WTA(8 h): 0.14 %. In the category of ‘Other’, we found that most of assignment could be considered as artificial alignments (chicken, rabbit, fruit fly, vector, etc.) which might be attributed to sequence homology. Undefined: failed to be aligned. (DOCX 130 kb)

The coefficient of variation for whole genome and each segment. (DOCX 18 kb)

The coefficient of variation for whole genome and each segment. (DOCX 18 kb)