I am trying to map single-cell chromatin accessibility data from doi:10.1038/nature14590, obtained using scATAC-seq, to the reference genome. Example paired-end reads are here.
The reads contain various adapter sequences, which the authors "trimmed from FASTQs using custom python scripts" [ref]. I am having problems reproducing this trimming. They used Nextera Transposase Adapters
and custom barcode adapters which are listed [here]. An excerpt from this list:
Custom Barcodes Adapter 1 (index i5): AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTAT AATGATACGGCGACCACCGAGATCTACACTAACCAAGTCGTCGGCAGCGTCAGATGTGTAT AATGATACGGCGACCACCGAGATCTACACAGGTTGCCTCGTCGGCAGCGTCAGATGTGTAT Custom Barcodes Adapter 2 (index i7): CAAGCAGAAGACGGCATACGAGATACCCAGCAGTCTCGTGGGCTCGGAGATGTG CAAGCAGAAGACGGCATACGAGATCCCAACCTGTCTCGTGGGCTCGGAGATGTG CAAGCAGAAGACGGCATACGAGATCACCACACGTCTCGTGGGCTCGGAGATGTG
Analyzing the FASTA-files with FastQC showed that the reverse complement of those sequences was over-represented in the data and also confirmed Nextera Transposase Adapters.
I tried to trim the sequences using
cutadapt --minimum-length=20 -a CACATCTCCGAGCCCACGAGAC -a CTGTCTCTTATA -A ATACACATCTGACGCTGCCGACGA -A CTGTCTCTTATA -o SRR1780164_1_trimmed.fastq.gz -p SRR1780164_2_trimmed.fastq.gz SRR1780164_1.fastq.gz SRR1780164_2.fastq.gz
CACATCTCCGAGCCCACGAGACbeing the reverse complement of the 2nd fix part of the i7 adapters
ATACACATCTGACGCTGCCGACGAbeing the reverse complement of the 2nd fix part of the i5 adapters
CTGTCTCTTATAfor the Nextera Transposase Adapters
FastQC does not indicate overrepresented sequences in the resulting FASTA-files, however still indicates Nextera Transposase Adapters in the _1 files. On the other hand, after TrimGalore removes
CTGTCTCTTATA, Nextra Adapters are not indicated anymore, for some reason.
Furthermore, mapping the reads using
bowtie2 -x GRCh38_primary_assembly_index -1 SRR1780164_1_trimmed.fastq.gz -2 SRR1780164_2_trimmed.fastq.gz > SRR1780164.sam
results in a low alignment rate
1352 (2.79%) aligned concordantly exactly 1 time
3420 (7.07%) aligned concordantly >1 times
11.25% overall alignment rate
which gives me the impression I did something wrong during adapter trimming.
Any help would be much appreciated.
Edit: As I am not sure about the sequences to use for trimming, I also tried
-a CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -A CTGTCTCTTATACACATCTGACGCTGCCGACGA, as found in a suggestion for Nextera Adapters, however it did not remove the sequence over-representation in the -1 file.
-a CACATCTCCGAGCCCACGAGAC -a CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -A CTGTCTCTTATACACATCTGACGCTGCCGACGA solves adapter-issues according to FastQC, but also does not give good mapping results.