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--- a +++ b/tests/test_demultiplexing.py @@ -0,0 +1,252 @@ +#!/usr/bin/env python3 +# -*- coding: utf-8 -*- +import unittest +from singlecellmultiomics.fastqProcessing.fastqIterator import FastqRecord +from singlecellmultiomics.barcodeFileParser.barcodeFileParser import BarcodeParser +from singlecellmultiomics.modularDemultiplexer.demultiplexModules.CELSeq2 import CELSeq2_c8_u6_NH +from singlecellmultiomics.modularDemultiplexer.demultiplexModules.scCHIC import SCCHIC_384w_c8_u3_cs2 +from singlecellmultiomics.modularDemultiplexer.demultiplexModules.DamID import DamID2andT_SCA,DamID2_SCA +from singlecellmultiomics.modularDemultiplexer.baseDemultiplexMethods import UmiBarcodeDemuxMethod +import pkg_resources +from singlecellmultiomics.utils import reverse_complement + +class TestUmiBarcodeDemux(unittest.TestCase): + + def test_UmiBarcodeDemuxMethod_matching_barcode(self): + + barcode_folder = pkg_resources.resource_filename('singlecellmultiomics','modularDemultiplexer/barcodes/') + index_folder = pkg_resources.resource_filename('singlecellmultiomics','modularDemultiplexer/indices/') + barcode_parser = BarcodeParser(barcode_folder, lazyLoad='*') + index_parser = BarcodeParser(index_folder, lazyLoad='*') + + r1 = FastqRecord( + '@NS500414:628:H7YVNBGXC:1:11101:15963:1046 1:N:0:GTGAAA', + 'ATCACACACTATAGTCATTCAGGAGCAGGTTCTTCAGGTTCCCTGTAGTTGTGTGGTTTTGAGTGAGTTTTTTAAT', + '+', + 'AAAAA#EEEEEEEEEEEAEEEEEEEAEEEEEEEEEEEEEEEEEE/EEEEEEEEEEEE/EEEEEEEEEEEEEEEEEE' + ) + r2 = FastqRecord( + '@NS500414:628:H7YVNBGXC:1:11101:15963:1046 2:N:0:GTGAAA', + 'ACCCCAGATCAACGTTGGACNTCNNCNTTNTNCTCNGCACCNNNNCNNNCTTATNCNNNANNNNNNNNNNTNNGN', + '+', + '6AAAAEEAEE/AEEEEEEEE#EE##<#6E#A#EEE#EAEEA####A###EE6EE#E###E##########E##A#' + ) + demux = UmiBarcodeDemuxMethod(umiRead=0, + umiStart=0, + umiLength=3, + barcodeRead=0, + barcodeStart=3, + barcodeLength=8, + barcodeFileParser=barcode_parser, + barcodeFileAlias='maya_384NLA', + indexFileParser=index_parser, + indexFileAlias='illumina_merged_ThruPlex48S_RP', + random_primer_read=None, + random_primer_length=6) + + demultiplexed_record = demux.demultiplex([r1,r2]) + # The barcode sequence is ACACACTA (first barcode) + self.assertEqual( demultiplexed_record[0].tags['BC'], 'ACACACTA') + self.assertEqual( demultiplexed_record[0].tags['bi'], 1) # 1 from version 0.1.12 + + + + def test_CS2_NH_matching_barcode(self): + + barcode_folder = pkg_resources.resource_filename('singlecellmultiomics','modularDemultiplexer/barcodes/') + index_folder = pkg_resources.resource_filename('singlecellmultiomics','modularDemultiplexer/indices/') + barcode_parser = BarcodeParser(barcode_folder, lazyLoad='*') + index_parser = BarcodeParser(index_folder, lazyLoad='*') + + seq = 'TATGAGCAATCACACACTATAGTCATTCAGGAGCAGGTTCTTCAGGTTCCCTGTAGTTGTGT' + r1 = FastqRecord( + '@NS500414:628:H7YVNBGXC:1:11101:15963:1046 1:N:0:GTGAAA', + f'ATAATATCTGGGCA{seq}', + '+', + 'AAAAA#EEEEEEEEEEEAEEEEEEEAEEEEEEEEEEEEEEEEEE/EEEEEEEEEEEE/EEEEEEEEEEEEEEEEEE' + ) + r2 = FastqRecord( + '@NS500414:628:H7YVNBGXC:1:11101:15963:1046 2:N:0:GTGAAA', + 'ACCCCAGATCAACGTTGGACNTCNNCNTTNTNCTCNGCACCNNNNCNNNCTTATNCNNNANNNNNNNNNNTNNGN', + '+', + '6AAAAEEAEE/AEEEEEEEE#EE##<#6E#A#EEE#EAEEA####A###EE6EE#E###E##########E##A#' + ) + demux = CELSeq2_c8_u6_NH( + barcodeFileParser=barcode_parser, + indexFileParser=index_parser) + + demultiplexed_record = demux.demultiplex([r1,r2]) + # The barcode sequence is ACACACTA (first barcode) + self.assertEqual( demultiplexed_record[0].tags['BC'], 'TCTGGGCA') + self.assertEqual( demultiplexed_record[0].tags['bi'], 55) + self.assertEqual( demultiplexed_record[0].tags['RX'], 'ATAATA') + self.assertEqual( demultiplexed_record[0].sequence, seq) + + def construct_tchic_read(self,crx,ccb,trx,tcb,mr,linker): + seq = f'{crx}{ccb}{linker}{trx}{tcb}TTTTTTTTTTTTTTTTTTTTT{mr}' + r1 = FastqRecord( + '@NS500414:628:H7YVNBGXC:1:11101:15963:1046 1:N:0:GTGAAA', + f'{seq}', + '+', + 'A'*len(seq) + ) + r2 = FastqRecord( + '@NS500414:628:H7YVNBGXC:1:11101:15963:1046 2:N:0:GTGAAA', + reverse_complement(seq), + '+', + 'A'*len(seq) + ) + return r1, r2 + + def test_TCHIC(self): + + barcode_folder = pkg_resources.resource_filename('singlecellmultiomics','modularDemultiplexer/barcodes/') + index_folder = pkg_resources.resource_filename('singlecellmultiomics','modularDemultiplexer/indices/') + barcode_parser = BarcodeParser(barcode_folder, lazyLoad='*',) + index_parser = BarcodeParser(index_folder, lazyLoad='*') + + crx = 'TAT' + ccb = 'TAAGTGCT' + trx = 'CTGTTG' + tcb = 'ACAGAAGC' + mr = 'TGAGAGAGAGAGAGAGAGAGAGAGC' + linker = 'TATC' + r1,r2 = self.construct_tchic_read(crx,ccb,trx,tcb,mr,linker) + + demux = SCCHIC_384w_c8_u3_cs2( + barcodeFileParser=barcode_parser, + indexFileParser=index_parser) + + demultiplexed_record = demux.demultiplex([r1,r2]) + # The barcode sequence is ACACACTA (first barcode) + self.assertEqual( demultiplexed_record[0].tags['BC'], ccb) + self.assertEqual( demultiplexed_record[0].tags['bi'], 225) + self.assertEqual( demultiplexed_record[0].tags['dt'], 'VASA') + self.assertEqual( demultiplexed_record[0].tags['RX'], crx) + self.assertEqual( demultiplexed_record[0].tags['rx'], trx) + self.assertEqual( demultiplexed_record[1].sequence, reverse_complement(mr)[:len(mr)-4]) + + + r1 = FastqRecord( + '@NS500414:628:H7YVNBGXC:1:11101:15963:1046 1:N:0:GTGAAA', + 'GGCGACGTCCTTCACTATAGGGAGTTCTACAGTTCGACGATCCTTAAATGGTGAGTTTTTTTTTTTTTTTTTTTTTTTGACCGACGGTCCCCCCGGGACCC', + '+', + 'A'*len('GGCGACGTCCTTCACTATAGGGAGTTCTACAGTTCGACGATCCTTAAATGGTGAGTTTTTTTTTTTTTTTTTTTTTTTGACCGACGGTCCCCCCGGGACCC') + ) + r2 = FastqRecord( + '@NS500414:628:H7YVNBGXC:1:11101:15963:1046 2:N:0:GTGAAA', + 'CGATCCTTAAATGGTGAGTTTTTTTTTTTTTTTTTTTTTTTGACCGACGGTCCCCCCGGGACCCGACGGCGCGACGACGCCCGGGGCGCACTGGGGACAGT', + '+', + 'A'*len('CGATCCTTAAATGGTGAGTTTTTTTTTTTTTTTTTTTTTTTGACCGACGGTCCCCCCGGGACCCGACGGCGCGACGACGCCCGGGGCGCACTGGGGACAGT') + ) + demultiplexed_record = demux.demultiplex([r1,r2]) + self.assertEqual( demultiplexed_record[0].tags['BC'], 'GACGTCCT') + self.assertEqual( demultiplexed_record[0].tags['bi'], 214) + self.assertEqual( demultiplexed_record[0].tags['dt'], 'VASA') + self.assertEqual( demultiplexed_record[0].tags['RX'], 'GGC') + self.assertEqual( demultiplexed_record[0].tags['rx'], 'CTTAAA') + + + def construct_read_pair(self, prefix, content): + seq = f'{prefix}{content}' + r1 = FastqRecord( + '@NS500414:628:H7YVNBGXC:1:11101:15963:1046 1:N:0:GTGAAA', + f'{seq}', + '+', + 'A'*len(seq) + ) + r2 = FastqRecord( + '@NS500414:628:H7YVNBGXC:1:11101:15963:1046 2:N:0:GTGAAA', + reverse_complement(seq), + '+', + 'A'*len(seq) + ) + return r1, r2 + + def test_DAMID(self): + + barcode_folder = pkg_resources.resource_filename('singlecellmultiomics','modularDemultiplexer/barcodes/') + index_folder = pkg_resources.resource_filename('singlecellmultiomics','modularDemultiplexer/indices/') + barcode_parser = BarcodeParser(barcode_folder, lazyLoad='*',) + index_parser = BarcodeParser(index_folder, lazyLoad='*') + + # First single cell format without overhang: + # EG: DamID2_BC_001 3-TGCA-3-TATG + + first_barcode, second_barcode = 'TGCA', 'TATG' + first_umi = 'ACT' + second_umi='CTC' + read_contents = 'rrrrrr' + r1,r2 = self.construct_read_pair(f'{first_umi}{first_barcode}{second_umi}{second_barcode}',read_contents) + + + damid_demux = DamID2_SCA(barcodeFileParser=barcode_parser, + second_barcode_len=4, + indexFileParser=index_parser, + barcode_alias='DamID2_scattered_8bp', + ) + + demultiplexed_record = damid_demux.demultiplex([r1,r2]) + self.assertEqual( demultiplexed_record[0].tags['BC'], first_barcode+second_barcode) + self.assertEqual( demultiplexed_record[0].tags['bi'], 1) + self.assertEqual( demultiplexed_record[0].tags['RX'], first_umi+second_umi) + self.assertEqual( demultiplexed_record[0].sequence, read_contents) + + combined_demux = DamID2andT_SCA( + barcodeFileParser=barcode_parser, + indexFileParser=index_parser) + + # The internal DamID demux of the combined protocol should return the same result + demultiplexed_record = combined_demux.damid_demux.demultiplex([r1,r2]) + self.assertEqual( demultiplexed_record[0].tags['BC'], first_barcode+second_barcode) + self.assertEqual( demultiplexed_record[0].tags['bi'], 1) + self.assertEqual( demultiplexed_record[0].tags['RX'], first_umi+second_umi) + self.assertEqual( demultiplexed_record[0].sequence, read_contents) + + + demultiplexed_record = combined_demux.demultiplex([r1,r2]) + self.assertEqual( demultiplexed_record[0].tags['BC'], first_barcode+second_barcode) + self.assertEqual( demultiplexed_record[0].tags['bi'], 1) + self.assertEqual( demultiplexed_record[0].tags['dt'], 'DamID') + self.assertEqual( demultiplexed_record[0].tags['RX'], first_umi+second_umi) + self.assertEqual( demultiplexed_record[0].sequence, read_contents) + + + def test_3DEC_UmiBarcodeDemuxMethod_matching_barcode(self): + + barcode_folder = pkg_resources.resource_filename('singlecellmultiomics','modularDemultiplexer/barcodes/') + barcode_parser = BarcodeParser(barcode_folder,lazyLoad='*') + + r1 = FastqRecord( + '@Cluster_s_1_1101_1000', + 'ATCACACACTATAGTCATTCAGGAGCAGGTTCTTCAGGTTCCCTGTAGTTGTGTGGTTTTGAGTGAGTTTTTTAAT', + '+', + 'AAAAA#EEEEEEEEEEEAEEEEEEEAEEEEEEEEEEEEEEEEEE/EEEEEEEEEEEE/EEEEEEEEEEEEEEEEEE' + ) + r2 = FastqRecord( + '@Cluster_s_1_1101_1002', + 'ACCCCAGATCAACGTTGGACNTCNNCNTTNTNCTCNGCACCNNNNCNNNCTTATNCNNNANNNNNNNNNNTNNGN', + '+', + '6AAAAEEAEE/AEEEEEEEE#EE##<#6E#A#EEE#EAEEA####A###EE6EE#E###E##########E##A#' + ) + demux = UmiBarcodeDemuxMethod(umiRead=0, + umiStart=0, + umiLength=3, + barcodeRead=0, + barcodeStart=3, + barcodeLength=8, + barcodeFileParser=barcode_parser, + barcodeFileAlias='maya_384NLA', + indexFileParser=None, + indexFileAlias='illumina_merged_ThruPlex48S_RP', + random_primer_read=None, + random_primer_length=6) + + demultiplexed_record = demux.demultiplex([r1,r2]) + # The barcode sequence is ACACACTA (first barcode) + self.assertEqual( demultiplexed_record[0].tags['BC'], 'ACACACTA') + self.assertEqual( demultiplexed_record[0].tags['bi'], 1) + + +if __name__ == '__main__': + unittest.main()