Adapter trimming directions for NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina

Paired-end reads

For adapter trimming, we recommend Flexbar v3.3.0 or later, an open-source tool. To download Flexbar and for program usage, please visit the Flexbar GitHub page. Once Flexbar is installed, adapters can be trimmed with the following 2-step command.

flexbar --reads input_reads_1.fastq \
        --reads2 input_reads_2.fastq \
        --stdout-reads \
        --adapters tso_g_wo_hp.fasta \
        --adapter-trim-end LEFT \
        --adapter-revcomp ON \
        --adapter-revcomp-end RIGHT \
        --htrim-left GT \
        --htrim-right CA \
        --htrim-min-length 3 \
        --htrim-max-length 5 \
        --htrim-max-first \
        --htrim-adapter \
        --min-read-length 2 \
        --threads 1 | \
    flexbar \
        --reads - \
        --interleaved \
        --target output_reads \
        --adapters ilmn_20_2_seqs.fasta \
        --adapter-trim-end RIGHT \
        --min-read-length 2 \
        --threads 1

Make sure there are no extra characters after \ at the end of the line, when the above command is used in the shell.

The input files for this command are the paired-end sequencing reads from the sequencing instrument: input_reads_1.fastq and input_reads_2.fastq. The adapter files tso_g_wo_hp.fasta and ilmn_20_2_seqs.fasta, as well as example fastq input files with a small number of reads can be downloaded here.

Flexbar is executed in two steps: (1) trimming template-switching oligo-related sequences (tso_g_wo_hp.fasta), and (2) trimming Illumina adapters (ilmn_20_2_seqs.fasta). The trimmed output reads are: output_reads_1.fastq and output_reads_2.fastq. To shorten runtime and reduce disk usage, the two flexbar commands are connected by a pipe, avoiding writing intermediate files. Log files are written into flexbarOut.log and output_reads.log for steps (1) and (2), correspondingly.

Homopolymers adjacent to the template-switching oligo are trimmed as well, as specified by --htrim* options. G and T homopolymers are trimmed (--htrim-left GT --htrim-right CA). Homopolymer length to trim is 3-5 for G, and 3 or higher for T (--htrim-min-length 3 --htrim-max-length 5 --htrim-max-first).

Keeping a short minimum read length after trimming (--min-read-length 2) keeps informative long reads, whose mates may be short after trimming. The default parameter --threads 1 can be adjusted to a higher number depending on your machine.

In our tests, trimming with flexbar takes 1-2 minutes for 1 million 2x75 read pairs input fastq files on an m4.2xlarge AWS instance (8 core, 32 GB RAM, Intel Xeon E5-2676 v3 processor, 2.4 GHz), using --threads 4.

Single-end reads

The library prep kit and the adapter trimming method were developed and tested using paired-end reads. We therefore recommend paired-end, rather than single-end sequencing. The directions below are listed solely as a convenience for the users who, for whatever reason, choose to do single-end sequencing, and should not be taken as a recommendation to do single-end sequencing with this library prep kit. These directions have been tested less extensively than the directions for paired-end reads (above).

flexbar --reads test_input_reads_1.fastq \
        --stdout-reads \
        --adapters tso_g_wo_hp.fasta \
        --adapter-trim-end LEFT \
        --adapter-revcomp ON \
        --adapter-revcomp-end RIGHT \
        --htrim-left GT \
        --htrim-right CA \
        --htrim-min-length 3 \
        --htrim-max-length 5 \
        --htrim-max-first \
        --htrim-adapter \
        --min-read-length 2 \
        --threads 1 | \
    flexbar \
        --reads - \
        --target output_reads \
        --adapters ilmn_20_2_seqs.fasta \
        --adapter-trim-end RIGHT \
        --min-read-length 2 \
        --threads 1

Note that the single-end commands are identical to the paired-end ones, except they omit these 2 lines:

        --reads2 input_reads_2.fastq \

and

        --interleaved \