Example Amplicon Analysis

Abstract

This pipeline output recapitulates analysis described in AIC Donaldson, et al. 2025. Aging Gut-Brain Interactions: Pro-Inflammatory Gut Bacteria Are Elevated in Fecal Samples from Individuals Living with Alzheimer's Dementia. Geriatrics (Basel) 10:37 (PMID: 40126287)

Before initial amplicon analysis, cutadapt (M Martin, EMBnet J v17 2011 DOI) was used to trim reads. Trimmed sequences included Illumina adapters, primers (read1: 5' AGMGTTYGATYMTGGCTCAG 3' and read2: 5' GCTGCCTCCCGTAGGAGT 3') and any Ns at either end of both reads.

Initial amplicon analysis was performed using the dada2 package (Callahan et al., Nature Methods 2016, PMID: 27214047 ) in R.

Read1s and Read2s were truncated at 200 and 225 respectively, or at the first occurrence of quality less than or equal to 2 or 2 respectively. Thereafter pairs were discarded if cumulative errors along a read, as determined from quality values, were 2 or 2 respectively. Read pairs determined to be derived from PhiX DNA were removed as well.

Error models were computed from filtered sequences separately for Read1s and Read2s. After de-replication of filtered reads, error models were applied to determine putative de-noised Read1 and Read2 sequences, which were then merged into single amplicon sequence variants (ASVs). ASVs 0 to 600 bases in length were retained. Very long or short ASVs can result from non-specific priming.

After removal of putatively chimeric reads, taxonomy was assigned using silva_nr_v138_train_set.fa.gz to assign taxa to genus and silva_species_assignment_v138.fa.gz to assign species level.

Read sequence quality plots

Figure 1 - Base quality values summarised by position for Read1 (A) and Read2 (B). Green and orange continuous lines represents mean and median quality, respectively. Dashed orange lines represent the 25th and 75th percentiles of sequence quality. Red trace represents coverage. These plots reproduced in supplementary file 1_quality_profiles.pdf

Error models

## 111294200 total bases in 556471 reads from 5 samples will be used for learning the error rates.
## 103420350 total bases in 459646 reads from 4 samples will be used for learning the error rates.

## Read1 error convergence:  47, 0.44, 0.025, 0.0038, 0.00027, 0

## Read2 error convergence:  50, 0.55, 0.038, 0.0061, 0.0029, 0

Figure 2 - log10 error probability vs quality score for each observed base difference in Read1 (A) and Read2 (B). Expected trend shown in red. Fitted line shown in black.

Amplicon sequence variant (ASV) length analysis

Figure 3 - ASV lengths plotted as a histogram. See supplementary file 5_reads_per_seqlen.pdf for this histogram

0 of 36835 ASVs were removed that were either <0 or >600 bases long.

29584 of 36835 putatively chimeric ASVs were removed.

Tracking table of reads/ASVs at different stages of the analysis

	Input	Filtered	DenoisedR1	DenoisedR2	Merged	NoChimera
A001-1	245617	151980	150535	151076	146706	136105
A001-2	261367	165102	163369	164155	159042	146309
A002-1	143686	87940	87460	87641	86063	85453
A002-2	88526	54624	53946	54290	52074	51756
A003-1	159341	96825	96053	96486	94318	92912
A004-1	110850	67240	66956	67025	64893	64717
A004-2	295158	177173	175603	176293	167402	153553
A046-1	181781	112119	109238	110630	103597	89515
A046-2	184956	113073	110509	111505	105484	95749
B015-1	264369	162295	160697	161388	155894	149718
B015-2	225185	134128	133051	133495	129105	123808
B016-1	240872	153419	151392	152248	145309	128386
B016-2	317736	200209	197021	198288	184595	156334
C007-1	49382	31049	30338	30594	28643	27148
D008-1	111923	59358	57490	58167	53143	47190
D008-2	243310	137407	134062	134919	125485	106735
D010-1	230518	143817	141310	142517	133011	119077
D010-2	240750	156020	153816	154789	145279	129642
D011-1	125207	78600	77811	78069	75711	73476
D011-2	259237	152757	149542	151103	142104	132907
D012-1	209680	128346	124512	126131	113280	102929
D012-2	189598	119607	117671	118343	110734	103891
D018-1	243025	151461	150597	150845	145409	133595
D018-2	274818	163443	162032	162396	152300	130040
E013-1	179601	110316	106814	108469	97979	85915
E013-2	64290	39180	38616	38603	36740	36068
E014-1	176920	103323	100944	102018	95921	89943
E014-2	66745	43205	42317	42589	40474	39309
F017-2	82760	48442	47127	47623	44727	42763
F024-1	246471	158846	155982	157012	149377	133868
F024-2	207399	127617	124167	125308	116673	101892
G021-1	251268	162778	159541	160671	150716	130660
G021-2	191024	119075	116642	117756	111290	101468
G022-1	250753	149921	147129	148303	141386	124914
G022-2	193428	113591	111450	112154	107239	96486
G025-1	158840	96751	94862	95675	89472	81015
G025-2	189929	121341	119581	120460	115378	108466
H026-1	221197	136921	135976	136215	130779	118407
H026-2	264901	167227	166131	166373	159388	134786
I029-1	228039	141249	139804	140532	136294	129762
I030-1	192717	118940	117347	118078	112314	108335
I031-1	129690	78518	78058	78141	75411	75104
I031-2	300876	188610	184511	186548	173418	144685
I032-1	127471	76336	74488	75248	70478	67662
I033-1	312087	193144	189305	191115	178055	160202
I033-2	254404	158340	155886	157292	149125	131498
I034-1	239829	144659	142235	143097	132683	116118
J036-1	146798	85683	83400	84517	79159	76322
J037-1	178776	108624	106556	107555	101932	99024
J038-1	207682	128120	126401	127182	122620	117361
J039-1	86261	55138	53387	54185	49552	45884
J040-1	153073	97379	95299	96362	90358	86709
J040-2	242836	152338	149401	150836	142032	134337
K043-1	244709	153289	151805	152427	145794	136308
M049-1	214579	136628	133805	134971	127906	113029
M049-2	284329	178271	174876	175891	168316	148692
M050-1	215724	131588	129212	130243	121200	106041
N052-1	199480	120894	118454	119404	109586	95256
N052-2	257783	159851	158715	159284	154204	151017
N053-1	50772	31803	31499	31571	30923	30542
N053-2	80362	50491	49305	49812	46355	43354
N054-1	192490	119667	118564	119130	115726	113016
N055-1	216655	135400	133296	134241	124477	114004
N055-2	279564	177989	175436	176580	165903	151225
N056-1	184723	115633	112905	113977	107975	95332
N056-2	245655	160874	157973	159353	152402	137301
N058-1	308145	188990	184784	187276	170974	153381
N058-2	225890	140075	139016	139533	135670	129676
N059-1	256408	159763	156054	157885	148119	132854
povestrainmix	230916	146960	146690	146525	141420	128410

This table is found in supplementary file 7_tracking_table.tsv

Non-chimeric ASV counts table is found in supplementary file 9_asvs.txt and accompanying taxon assignments are found in 9_taxa.txt. A transposed ASV counts table (ASVs in rows and samples in columns) is found in 6_seqtab_nochim_t.txt

The ASV counts table for positive control samples is found in 8_mock_asvs_raw.txt and accompanying taxon assignments are found in 8_mock_taxa_raw.txt

Library versions

## R version 4.5.0 (2025-04-11)
## Platform: x86_64-pc-linux-gnu
## Running under: Ubuntu 24.04.2 LTS
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## BLAS:   /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.12.0 
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