Assembled and found similar clone sequences.

[fookloy]

HI, i found some sequences that are totally similar but the Valignscore and Calignscore are different in the assemble output. My sequence should cover the FR1 to FR4.

Why each sequences are same to each other but one still couldn't well align to the C region?

It seems OseparateByC=true might be the key parameter account for that, could you please tell how to adjust it?

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cloneId	readCount	readFraction	targetSequences	targetQualities	allVHitsWithScore	allDHitsWithScore	allJHitsWithScore	allCHitsWithScore	allVAlignments	allDAlignments	allJAlignments	allCAlignments	nSeqFR1	minQualFR1	nSeqCDR1	minQualCDR1	nSeqFR2	minQualFR2	nSeqCDR2	minQualCDR2	nSeqFR3	minQualFR3	nSeqCDR3	minQualCDR3	nSeqFR4	minQualFR4	aaSeqFR1	aaSeqCDR1	aaSeqFR2	aaSeqCDR2	aaSeqFR3	aaSeqCDR3	aaSeqFR4	refPoints
140	141	1.82E-04	CAGGTGCAGCTGCAGGAGTCCGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGGGTATAGCAGTGGCTGGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG	[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[	IGHV6-1*00(2804.9)	IGHD6-19*00(85)	IGHJ4*00(430)		80|384|405|0|304|SA85GSC95GSA100C|2953.0	22|39|63|304|321||85.0	25|68|68|321|364||430.0		CAGGTGCAGCTGCAGGAGTCCGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC	58	GGGGACAGTGTCTCTAGCAACAGTGCTGCT	58	TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG	58	ACATACTACAGGTCCAAGTGGTATAAT	58	GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTAC	58	TGTGCAAGAGGGTATAGCAGTGGCTGGTTTGACTACTGG	58	GGCCAGGGAACCCTGGTCACCGTCTCCTCAG	58	QVQLQESGPGLVKPSQTLSLTCAIS	GDSVSSNSAA	WNWIRQSPSRGLEWLGR	TYYRSKWYN	DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYY	CARGYSSGWFDYW	GQGTLVTVSS_	::::0:75:105:156:183:294:-1:304:304:-1:-3:321:321:-5:333:364::
1308	26	3.36E-05	CAGGTGCAGCTGCAGGAGTCCGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGGGTATAGCAGTGGCTGGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG	[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[	IGHV6-1*00(2804.2)	IGHD6-19*00(85)	IGHJ4*00(430)	IGHM*00(40.1)	80|384|405|0|304|SA85GSC95GSA100C|2953.0	22|39|63|304|321||85.0	25|68|68|321|364||430.0		CAGGTGCAGCTGCAGGAGTCCGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC	58	GGGGACAGTGTCTCTAGCAACAGTGCTGCT	58	TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG	58	ACATACTACAGGTCCAAGTGGTATAAT	58	GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTAC	58	TGTGCAAGAGGGTATAGCAGTGGCTGGTTTGACTACTGG	58	GGCCAGGGAACCCTGGTCACCGTCTCCTCAG	58	QVQLQESGPGLVKPSQTLSLTCAIS	GDSVSSNSAA	WNWIRQSPSRGLEWLGR	TYYRSKWYN	DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYY	CARGYSSGWFDYW	GQGTLVTVSS_	::::0:75:105:156:183:294:-1:304:304:-1:-3:321:321:-5:333:364::
1092	30	3.87E-05	CAGGTGCAGCTGCAGGAGTCCGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAAGGGGATAGCAGTGGCTGGTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG	[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[	IGHV6-1*00(2773.6)	IGHD6-19*00(81)	IGHJ4*00(420)	IGHM*00(40.9)	80|381|405|0|301|SA85GSC95GSA100C|2923.0	21|40|63|302|321|ST24G|81.0	26|68|68|322|364||420.0		CAGGTGCAGCTGCAGGAGTCCGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC	58	GGGGACAGTGTCTCTAGCAACAGTGCTGCT	58	TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG	58	ACATACTACAGGTCCAAGTGGTATAAT	58	GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTAC	58	TGTGCAAAGGGGATAGCAGTGGCTGGTCTTGACTACTGG	58	GGCCAGGGAACCCTGGTCACCGTCTCCTCAG	58	QVQLQESGPGLVKPSQTLSLTCAIS	GDSVSSNSAA	WNWIRQSPSRGLEWLGR	TYYRSKWYN	DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYY	CAKGIAVAGLDYW	GQGTLVTVSS_	::::0:75:105:156:183:294:-4:301:302:0:-2:321:322:-6:333:364::
88	210	2.71E-04	CAGGTGCAGCTGCAGGAGTCCGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAAGGGGATAGCAGTGGCTGGTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG	[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[	IGHV6-1*00(2774.7)	IGHD6-19*00(81)	IGHJ4*00(420)		80|381|405|0|301|SA85GSC95GSA100C|2923.0	21|40|63|302|321|ST24G|81.0	26|68|68|322|364||420.0		CAGGTGCAGCTGCAGGAGTCCGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC	58	GGGGACAGTGTCTCTAGCAACAGTGCTGCT	58	TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG	58	ACATACTACAGGTCCAAGTGGTATAAT	58	GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTAC	58	TGTGCAAAGGGGATAGCAGTGGCTGGTCTTGACTACTGG	58	GGCCAGGGAACCCTGGTCACCGTCTCCTCAG	58	QVQLQESGPGLVKPSQTLSLTCAIS	GDSVSSNSAA	WNWIRQSPSRGLEWLGR	TYYRSKWYN	DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYY	CAKGIAVAGLDYW	GQGTLVTVSS_	::::0:75:105:156:183:294:-4:301:302:0:-2:321:322:-6:333:364::
1069	31	4.00E-05	CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAAGGGGATAGCAGTGGCTGGTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG	[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[	IGHV6-1*00(2862)	IGHD6-19*00(81)	IGHJ4*00(420)	IGHM*00(40.5)	80|381|405|0|301||3010.0	21|40|63|302|321|ST24G|81.0	26|68|68|322|364||420.0		CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC	58	GGGGACAGTGTCTCTAGCAACAGTGCTGCT	58	TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG	58	ACATACTACAGGTCCAAGTGGTATAAT	58	GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTAC	58	TGTGCAAAGGGGATAGCAGTGGCTGGTCTTGACTACTGG	58	GGCCAGGGAACCCTGGTCACCGTCTCCTCAG	58	QVQLQQSGPGLVKPSQTLSLTCAIS	GDSVSSNSAA	WNWIRQSPSRGLEWLGR	TYYRSKWYN	DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYY	CAKGIAVAGLDYW	GQGTLVTVSS_	::::0:75:105:156:183:294:-4:301:302:0:-2:321:322:-6:333:364::
67	247	3.19E-04	CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAAGGGGATAGCAGTGGCTGGTCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG	[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[	IGHV6-1*00(2861.9)	IGHD6-19*00(81)	IGHJ4*00(420)		80|381|405|0|301||3010.0	21|40|63|302|321|ST24G|81.0	26|68|68|322|364||420.0		CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC	58	GGGGACAGTGTCTCTAGCAACAGTGCTGCT	58	TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG	58	ACATACTACAGGTCCAAGTGGTATAAT	58	GATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTAC	58	TGTGCAAAGGGGATAGCAGTGGCTGGTCTTGACTACTGG	58	GGCCAGGGAACCCTGGTCACCGTCTCCTCAG	58	QVQLQQSGPGLVKPSQTLSLTCAIS	GDSVSSNSAA	WNWIRQSPSRGLEWLGR	TYYRSKWYN	DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYY	CAKGIAVAGLDYW	GQGTLVTVSS_	::::0:75:105:156:183:294:-4:301:302:0:-2:321:322:-6:333:364::

nohup java -Xmx100g -jar /mnt/C_NGS01/Fred/antibody/mixcr.jar align --preset generic-amplicon --species hsa --rna --rigid-left-alignment-boundary --rigid-right-alignment-boundary C -OmergerParameters.minimalOverlap=10 -OmergerParameters.minimalIdentity=0.9 ${id}_1.fq.gz ${id}_2.fq.gz ${id}_align.vdjca

nohup java -Xmx100g -jar /mnt/C_NGS01/Fred/antibody/mixcr.jar assemble --assemble-clonotypes-by "{FR1Begin:FR4End}" -OseparateByC=true -ObadQualityThreshold=0 HH-hH_L2_align.vdjca HH-hH_L2_assemble.clns &

[mizraelson]

It's hard to tell without looking at the alignments what went wrong. It could be that only a short C gene sequence is covered, and in some reads, there is a mismatch in that position, which brings the score down, and MiXCR does not identify it with the default thresholds. You can change the parameter to:

-OseparateByC=false

This way, MiXCR will not split clones depending on the C gene if the VDJ region is identical.