Oxford Nanopore Duplex data for HG002. Duplex reads are found in the stereo_duplex and additional_duplex folders.
For questions about this data, contact khmiga@ucsc.edu

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##                                 Layout                                    ##
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stereo_duplex/
	*.bam  (All reads)
	*_pass.bam (reads over Q10)
	*_pass.fastq.gz (reads over Q10)
	*_fail.bam (reads under Q10)
	*_fail.fastq.gz (reads under Q10)
additional_duplex/
	*.bam  (All reads)
	*_pass.bam (reads over Q10)
	*_pass.fastq.gz (reads over Q10)
	*_fail.bam (reads under Q10)
	*_fail.fastq.gz (reads under Q10)
simplex/
	*.bam (used for duplex calling, not recommended to use as inputs to analysis)
	*.fastq.gz (used for duplex calling, not recommended to use as inputs to analysis)
ancillary_files/
	pair_ids/ (used for duplex calling, not recommended to use as inputs to analysis)
	sequencing_summary/ (used for duplex calling, not recommended to use as inputs to analysis)


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##                               Data Generation                             ##
###############################################################################

## Simplex calling

Fast5 files were converted to POD5 and then grouped by channel with:
```
pod5 convert fast5 --force-overwrite --threads 90 ${FAST5}/*.fast5 ${POD5}/output.pod5
pod5 subset --force_overwrite --output ${POD5_GROUPED} --summary $SEQSUMMARY --columns $POD5_GROUPING -M ${POD5}/output.pod5
```

Call Simplex data with Dorado:
```
MODEL_PATH="dorado_v4_duplex_beta_models/dna_r10.4.1_e8.2_400bps_sup@v4.0.0"
dorado basecaller -x "cuda:all" $MODEL_PATH $POD5_GROUPED > ${OUTPUT}/${output_name}_Dorado_v0.1.1_400bps_sup.sam
```

Converted from sam to fastq.gz and bam with samtools


## Duplex calling:
```
duplex_tools pair ${OUTPUT}/${output_name}_Dorado_v0.1.1_400bps_sup.bam
dorado duplex ${MODEL_PATH} $POD5_GROUPED --pairs ${OUTPUT}/pairs_from_bam/pair_ids_filtered.txt > ${OUTPUT}/${output_name}_Dorado_v0.1.1_400bps_sup_stereo_duplex.sam
```
convert to bam with samtools and Pass/Fail files are generated by filtering for reads by Q-score


## Read rescue and duplex calling on rescued reads:
​
For extra duplex, first fast-call (with --emit-moves) 
```
FAST_MODEL_PATH="dorado_v4_duplex_beta_models/dna_r10.4.1_e8.2_400bps_fast@v4.0.0"
dorado basecaller ${FAST_MODEL_PATH} ${POD5} --emit-moves > ${OUTPUT}/${output_name}_unmapped_reads_with_moves.sam
​```

Second, use duplex tools split pairs to recover non-split duplex reads
```
duplex_tools split_pairs ${OUTPUT}/${output_name}_unmapped_reads_with_moves.sam ${POD5} pod5s_splitduplex/
​```
Finally, duplex-call with sup
```
dorado duplex ${MODEL_PATH} pod5s_splitduplex/ --pairs split_duplex_pair_ids.txt > ${OUTPUT}/${output_name}_duplex_splitduplex.sam
```
convert to bam with samtools and Pass/Fail files are generated by filtering for reads by Q-score